JP2014511680A - Bispecific anti-CXCR7 immunoglobulin single variable domain - Google Patents

Abstract

The present invention relates to specific polypeptides, nucleic acids encoding such polypeptides; methods for preparing such polypeptides; host cells that express or are capable of expressing such polypeptides. And to compositions, and in particular to pharmaceutical compositions containing such polypeptides for prophylactic, therapeutic or diagnostic purposes. In particular, the present invention provides immunoglobulin single variable domains that inhibit CXCR7-mediated tumor growth.

Description

The present invention relates to biological materials and polypeptides, methods related to CXCR7 comprising nucleic acids encoding such polypeptides; methods for preparing such polypeptides; such A host cell that expresses or is capable of expressing a polypeptide; for example, a composition comprising a pharmaceutical composition comprising such a polypeptide, such as for prophylactic, therapeutic, or diagnostic purposes .

BACKGROUND OF THE INVENTION In the art, i) CXCR7 blockade used in conjunction with CXCR4 blockade may be useful for the treatment of SDF-1-dependent tumor progression and metastasis (RB Maksym et al., 2009, The role of stromal-derived factor-1-CXCR7 axis in development of cancer, European Journal of Pharmacology, 625 (1-3), pages 31-40) and ii) some small molecule inhibitors such as CCX733 or CCX266, siRNA and Blocking antibodies (clone Mab 11G8, Mab 9C4, see eg US20070167443; clone 358426 (R & D Systems); Mab 8F11 (Biolegend)), etc. for therapeutic interference with CXCR4-mediated activation of integrins Can be useful. (TN Hartmann et al., 2008, A crosstalk between intracellular CXCR7 and CXCR4 involved in rapid CXCL12-triggered integrin activation but not in chemokine-triggered motility of human T lymphocytes and CD34 + cells, Journal of Leukocyte Biology, 84, pages 11301140) However, the biology of CXCR7 is still poorly understood. This is because the mechanism of action of CXCR7 is unknown. Because i) it can act as a kind of decoy or signaling receptor, depending on cell type (RM Maksym et al., Supra) and ii) SDF to I-TAC and CXCR7 This is because the interaction between -1 is unknown.

  The identification of selective therapeutically effective anti-CXCR7 drugs is not possible with their poorly understood biology (eg, the mechanism of action of potential agonist CCX733 or CCX266 versus antagonist, interaction with CXCR4 Not only because of the recognition of important epitopes, cross-reactivity of compounds CCX733 or CCX266, and related toxicities, but also in the art (see, eg, Naunyn-Schmied Archives Pharmacology 379: 385-388) ), And the production of anti-GPCR therapeutic agents (such as anti-CXCR7 agents) has been recognized as difficult. Because i) the natural conformation of active CXCR7 in cancer cells is not strictly known, and ii) CXCR7 is less immunogenic (and also exposed to highly conserved extracellular surfaces) This is due to the limited number of residues, eg, mouse-human CXCR7 is predicted to show 96% homology).

  In addition, compounds (CCX733, CCX754), they can selectively block the binding of CXCL11 and CXCL12 to CXCR7 and function like chemokine ligands with respect to homodimerization, ie they are CXCR7 Homodimerization was enhanced by 2.5 and 3.5 fold, and significant increases (P <0.05) were first detected at 10 and 100 nM (KE Luker et al., 2009, Imaging chemokine receptor dimerization with firefly luciferase complementation, FASEB journal, 23, pages 823-834).

  CXCR7 is due to a potential role in tumor development. Because its expression provides cells with growth and survival benefits. Recently, CXCR7 has been demonstrated to promote breast and lung tumor growth and enhance lung metastasis (Proc. Natl. Acad. Sci. USA 2007 104: 15735-15740). Furthermore, CXCR7 expression correlates with tumor aggressiveness in prostate cancer (J. Biol. Chem 2008 283: 4283-4294). Administration of small molecule antagonists to CXCR7 results in interference with tumor growth in animal models and validates CXCR7 as a target for the development of new cancer therapeutics (J. Exp. Med. 2006 203: 2201-2213 ).

  Head and neck cancer is among the most prevalent tumors in the world. Despite advances in the treatment of head and neck tumors, the survival of patients with these cancers has improved significantly over the past decades due to the inability to control local and distant metastases of the disease and poor understanding. Not. Head and neck cancer is consistently ranked among the six most frequently diagnosed cancers in the world. Oral and pharyngeal cancer alone accounts for about 300,000 new cases and just under 200,000 deaths annually worldwide. Over 90% of head and neck cancers are squamous cell carcinomas of the upper respiratory tract (including the oral cavity, pharynx, larynx, and sinuses). Epithelial head and neck tumors can also occur in the salivary and thyroid glands. Despite advances in our understanding of head and neck cancer and advances in its prevention and treatment, the survival of patients with head and neck cancer has not been significantly improved over the past decades.

SUMMARY OF THE INVENTION Both WO 2006/116319 and WO 2008/048519 describe the production of antibodies to G protein coupled receptors (GPCRs) as notorious and difficult. Indeed, the production of conventional anti-CXCR7 antibodies has been described only in a limited number of cases, eg in WO 2006/116319 for conventional antibodies 11G8, 6E10 and in Zabel et al. For conventional antibody 8F11. (Zabel et al., 2009, Elucidation of CXCR7 mediated signaling events and inhibition of CXCR4 mediated tumor cell transendothelial migration by CXCR7 ligands. J Immunol .; 183 (5): 3204-11). However, despite extensive research, it is currently unclear whether these or similar antibodies are suitable for medical applications.

  Zheng et al. Reported increased CXCR7 expression in hepatocellular carcinoma tissue. Down-regulation of CXCR7 expression results in reduced tumor growth in a HCC xenograft model. However, the authors used SMMC-7721 cells, which were previously transfected in vitro with CXCR7 shRNA (Zheng et al. 2010 "Chemokine receptor CXCR7 regulates the invasion, angiogenesis and tumour growth of human hepatocellular carcinoma cells. "J. Exp. Clin. Cancer Res. 29:31).

  Small molecules are known for side effects and unwanted effects. Small molecule CCX771 blocks CXCL12 binding (see Carbajal et al; 2010 "Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis Proc Natl Acad Sci USA. 107: 11068-11073) On the other hand, it has been described as a synthetic CXCR7 ligand CCX771, which also potently stimulates β-arrestin mobilization to CXCR7 with greater potency and efficacy than endogenous chemokine ligands (Zabel et al. 2009 "Elucidation of CXCR7-Mediated Signaling Events and Inhibition of CXCR4-Mediated Tumor Cell Transendothelial Migration by CXCR7 Ligands" J. Immun. 183: 0000-0000) Similarly, the small compound VUF11403 (VU Amsterdam) Behave as an agonist.

  Currently, there are no anti-CXCR7 drugs on the market or clinical.

  Thus, there is a need for potent anti-CXCR7 agents that can explore and establish the medical potential of this target. Further, there is a need for anti-CXCR7 agents (eg, those provided herein) that are diagnostically, preventatively and / or therapeutically suitable.

  CXCR7 is expressed on many human tumor cells (but not on most healthy cells). In our tumor model system, we found that reduction or inhibition of CXCR7 by a single variable domain of immunoglobulin reduces or eliminates tumor formation in vivo.

  Immunoglobulin sequences, such as antibodies and antigen-binding fragments derived from them (eg, single variable domains of immunoglobulins) are used to specifically target their respective antigens in research and therapeutic applications. Generation of immunoglobulin single variable domains (eg, VHH, etc.) can be achieved by immunizing laboratory animals (eg, llamas), building phage libraries from immune tissues, antigen-binding immunoglobulin single variable domains. Selection of phage to display, as well as screening of the domains and their engineered constructs for the desired specificity (WO 94/04678). Alternatively, selecting a single variable domain of an immunoglobulin (eg, a dAb, etc.), a phage that displays the single variable domain of an antigen-binding immunoglobulin directly from a natural or synthetic library, and Can be generated by screening the domains and their engineering constructs for the desired specificity (Ward et al, Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli, Nature, 1989, Oct 12; 341 ( 6242): 544-6); Holt et al., Trends Biotechnol., 2003, 21 (11): 484-490; and, for example, WO 06/030220, WO 06/003388 and other published patents of Domantis Ltd. application).

  Targeting serum albumin and extending the half-life of a biological molecule (eg, a single variable domain of an immunoglobulin, etc.) is described in, eg, WO 2008/028977.

  In one aspect, the invention provides: i) a first building block consisting essentially of a single variable domain of one or more immunoglobulins, wherein said single variable domain of immunoglobulins is against CXCR7 And ii) a second building block consisting essentially of a single variable domain of one or more (preferably one) immunoglobulins, wherein said The immunoglobulin single variable domains are directed against serum albumin, in particular against human serum albumin (and even more preferably, wherein the immunoglobulin single variable domain is Alb8 (as defined herein)))). Furthermore, the invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; host cells that express or are capable of expressing such polypeptides. In compositions, and in particular in pharmaceutical compositions comprising such polypeptides, nucleic acids, and / or host cells; and as such for prophylactic, therapeutic or diagnostic purposes The use of such polypeptides, nucleic acids, host cells, and / or compositions. Other aspects, embodiments, advantages and applications of the invention will become apparent from the further description herein.

FIG. 1 shows an SDF-1 competition experiment using FACS. FIG. 2 shows a Mab 11G8 competition experiment using FACS. FIG. 3 shows an immunohistochemical analysis of CXCR7 expression in sections of primary tumors. FIG. 4 shows CXCR7 mRNA profiling in head and neck cancer cell lines 11B, 22A, 22B, FaDu, OE, and 93-VU-147 by qPCR. FIG. 5 shows ligands CXCL11, CXCL12, Nanobody 09A04, and negative control: no competitor (named by “-”, indicating total binding (TB)); and CXCL10 (showing a-specific competition). [ ^125I ] -CXCL12 competition experiments with head and neck cancer cell lines 11B, 22A, 22B, FaDu, OE, and 93-VU-147 used. FIG. 6 shows in vivo CXCR7 Nanobody treatment with 22A grafts in nude mice: “−” negative control (PBS); polypeptide construct clone 060, clone 083, clone 085, and clone 093. FIG. 7 shows tumor volume after 50 days of treatment with in vivo CXCR7 Nanobody therapy with 22A grafts in nude mice: “−” negative control (PBS); polypeptide construct clone 085 and clone 093. FIG. 8 shows inhibition of SDF-1 binding to HEK293T hCXCR7 in the presence of 2 mg / ml HSA.

Definition of description of invention:
a) Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art and will be apparent to those skilled in the art. References are made, for example, to the standard handbook mentioned in paragraph a) of page 46 of WO 08/020079.

b) Unless otherwise indicated, the term “immunoglobulin single variable domain” (ISVD) is used as a general term, but is not limited to antigen binding domains or fragments (eg, V _HH domains or V _H Or _VL domain). The terms antigen binding molecule or antigen binding protein are used interchangeably and also include the term Nanobody. The single variable domain of an immunoglobulin is further a light chain variable domain sequence (eg, _VL sequence) or a heavy chain variable domain sequence (eg, V _H sequence); more specifically, they are conventional Or a heavy chain variable domain sequence derived from a heavy chain antibody. Thus, a single variable domain of an immunoglobulin is suitable for use as a domain antibody (or an immunoglobulin sequence suitable for use as a domain antibody), a single domain antibody (or as a single domain antibody) Immunoglobulin sequences), “dAbs” (or immunoglobulin sequences suitable for use as dAbs), or nanobodies (including but not limited to V _HH sequences). The present invention includes immunoglobulin sequences from different origins, including mouse, rat, rabbit, donkey, human, and camel immunoglobulin sequences. A single variable domain of an immunoglobulin includes fully human, humanized, other sequence optimized or chimeric immunoglobulin sequences. The structure of an immunoglobulin single variable domain and an immunoglobulin single variable domain, but without limitation, may be considered to consist of four framework regions or "FRs" Are referred to in the art and herein as “framework region 1” or “FR1”; as “framework region 2” or “FR2”; as “framework region 3” or “FR3”; Referred to as work region 4 "or" FR4 "respectively; those framework regions are interrupted by three complementarity determining regions or" CDRs ", which are referred to in the art as" complementarity determining regions 1 "or As “CDR1”; as “complementarity determining region 2” or “CDR2”; and “complementarity determining region 3” or “ It is referred to respectively as DR3 ". The term nanobody or nanobody is a registered trademark of Ablynx NV and is described as such and may also be referred to as Nanobody® and / or Nanobody®.

c) Unless otherwise indicated, the terms “immunoglobulin sequence”, “sequence” “nucleotide sequence” and “nucleic acid” are as described in paragraph b) of page 46 of WO 08/020079. The term Nanobody is also as defined in WO 08/020079 and, as described herein, generally V _HH domains (eg, V _H from “heavy chain only” antibodies that occur in camelids). Domain) functional and / or structural characteristics of immunoglobulin heavy chain variable domains, such as (natural) V _HH , humanized V _HH or camelized V _H (eg camelized human) VH etc.).

  d) Unless otherwise indicated, all methods, steps, techniques and manipulations not specifically described in detail can be performed and are performed in a manner known per se and will be apparent to those skilled in the art. That's right. Reference is again made, for example, to the standard handbook and the general background art referred to herein, and to further references cited herein; and, for example, the following review, Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2 (1): 49-57; Irving et al., J. Immunol. Methods, 2001, 248 (1-2), 31-45; Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et al., Tumour Biol., 2005, 26 (1), 31-43 They describe techniques for protein manipulation, such as affinity maturation and other techniques to improve the specificity of proteins (such as immunoglobulins) and other desired properties.

  e) Amino acid residues may be indicated according to the standard 3 letter or 1 letter amino acid code. Reference is Ablynx NV International Application WO 08/020079, Table 48, page 48 of the title “Immunoglobulin single variable domains directed against IL-6R and comprising comprising the same for the treatment of diseases and disorders associated with Il-6 mediated signaling”. -2 is made.

  f) For purposes of comparing two or more nucleotide sequences, the percentage of “sequence identity” between the first and second nucleotide sequences is incorporated by reference in WO 08/020079 (herein incorporated by reference). ) May be calculated or determined as described in page 49, paragraph e), eg, [number of nucleotides in the first nucleotide sequence that are identical to the nucleotide at the corresponding position in the second nucleotide sequence. ] By [total number of nucleotides in the first nucleotide sequence] and multiplied by [100%], where each deletion, insertion, substitution, or addition of each nucleotide in the second nucleotide sequence is Considered as the difference in a single nucleotide (position) compared to the first nucleotide sequence; or And using a computer algorithm or technique, again as described in WO 08/020079 paragraph e page 49 (incorporated is herein by reference)).

  g) a “sequence between a first amino acid sequence and a second amino acid sequence for the purpose of comparing a single variable domain or other amino acid sequence of two or more immunoglobulins (eg, a polypeptide of the invention, etc.). The percentage of “identity” (also referred to herein as “amino acid identity”) is described in paragraphs f) of pages 49 and 50 of WO 08/020079 (incorporated herein by reference). For example, [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] is calculated as [in the first amino acid sequence] Divided by [total number of amino acids] and multiplied by [100%], where each deletion, insertion, and substitution of each amino acid residue in the second amino acid sequence Or addition is considered as a difference at a single amino acid residue (position) compared to the first amino acid sequence, ie, as an “amino acid difference” as defined herein; or a suitable computer Again, as described in paragraph f) of pages 49 and 50 of WO 08/020079 (incorporated herein by reference) by using algorithms or techniques.

  Also, in determining the degree of sequence identity between two immunoglobulin single variable domains, one of skill in the art can take into account so-called “conservative” amino acid substitutions (described in page 50 of WO 08/020079). As you do).

Any amino acid substitutions that apply to the polypeptides described herein are also those of amino acid variation between different species of homologous proteins developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978. For the analysis of frequency, for the analysis of structures forming the potential developed by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and for Eisenberg et al., Proc. Natl. Acad Sci. USA 81: 140-144, 1984; Kyte &Doolittle; J Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353 , 1986 (all incorporated herein by reference in their entirety). Information regarding the primary, secondary, and tertiary structure of Nanobodies is given in the description herein and in the general background art cited above. For this purpose, the crystal structure of the V _HH domain from the llama is also described, for example, by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural Biology ( 1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). In the conventional V _H domains, VH / VL interface and potential camelizing substitutions additional information about some of the amino acid residues that form at these positions can be found in the prior art cited above.

  h) Single immunoglobulin variable domain and nucleic acid sequences are said to be “exactly the same” if they have 100% sequence identity (as defined herein) over their entire length.

  i) When comparing two immunoglobulin single variable domains, the term “amino acid difference” refers to the insertion, deletion, or deletion of a single amino acid residue at a position of the first sequence compared to the second sequence, or It is understood that a single variable domain of two immunoglobulins may contain one, two, or more such amino acid differences.

  j) When a nucleotide sequence or amino acid sequence “comprises” another nucleotide sequence or amino acid sequence, respectively, or “consists essentially of” another nucleotide sequence or amino acid sequence, this is defined in WO 08/020079. Has the meaning given in paragraph i) on pages 51-52.

  k) The term “in substantially isolated form” has the meaning given to it in paragraphs j) of pages 52 and 53 of WO 08/020079.

  l) The terms “domain” and “binding domain” have the meaning given to them in paragraph k) of page 53 of WO 08/020079.

  m) The terms “antigenic determinant” and “epitope” may also be used interchangeably herein and have the meaning given to them in paragraph l) of page 53 of WO 08/020079. Have.

  n) as described further in paragraph m) of page 53 of WO 08/020079, which can (specifically) bind and for a particular antigenic determinant, epitope, antigen or protein (or at least one thereof) Amino acid sequences (for example, antibodies, polypeptides of the invention, or, in general, antigen binding proteins or polypeptides or fragments thereof) that have affinity and / or specificity (for a portion, fragment, or epitope) Said antigenic determinant, epitope, antigen or protein is said to be “to” or “directed against”.

o) The term “specificity” has the meaning given to it in paragraph n) of pages 53-56 of WO 08/020079; and, as referred to herein, a specific antigen binding molecule or antigen binding protein Refers to the number of different types of antigens or antigenic determinants to which a molecule can bind (eg, a polypeptide of the invention). The specificity of an antigen binding protein can be determined based on affinity and / or binding power, as described on pages 53-56 of WO 08/020079 (incorporated herein by reference), Also described are some preferred techniques for measuring binding between an antigen-binding molecule (eg, a polypeptide of the invention) and related antigens. Typically, an antigen binding protein (such as a single variable domain and / or polypeptide of an immunoglobulin of the present invention) is 10 ⁻⁵ to 10 ⁻¹² mol / liter or less, and preferably With a dissociation constant (KD) of 10 ⁻⁷ to 10 ⁻¹² mol / liter or less, and preferably 10 ⁻⁸ to 10 ⁻¹² mol / liter (ie, 10 ⁵ to 10 ¹² l / mol or more, and preferably Can bind with an association constant (KA) of from 10 ⁷ to 10 ¹² liters / mole or more, and more preferably from 10 ⁸ to 10 ¹² liters / mole. Any KD value greater than 104 mol / liter (or any KA value less than 10 ⁴ M ⁻¹ ) liter / mol is generally considered to indicate non-specific binding. Preferably, a single variable domain of a monovalent immunoglobulin of the present invention binds to a desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of an antigen binding protein to an antigen or antigenic determinant can be achieved by any suitable manner known per se (eg, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (Such as EIA) and sandwich competition assays) and different variants thereof known per se in the art; and other techniques referred to herein. As will be apparent to those skilled in the art, and as described in pages 53-56 of WO 08/020079, the dissociation constant may be the actual or apparent dissociation constant. Methods for determining the dissociation constant will be apparent to those skilled in the art and include, for example, the techniques mentioned on pages 53-56 of WO 08/020079.

  p) The half-life of the amino acid sequences, compounds or polypeptides of the invention can generally be defined as described in paragraph o) of page 57 of WO 08/020079 and is referred to herein. As such, the serum concentration of an amino acid sequence, compound, or polypeptide is reduced by 50% in vivo, eg due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms Refers to the time required for this. The in vivo half-life of the amino acid sequences, compounds or polypeptides of the invention can be determined in any manner known per se, for example by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art, but can be generally as described, for example, in paragraph o) of page 57 of WO 08/020079. Also, as mentioned in paragraph o) of page 57 of WO 08/020079, half-life should be expressed using parameters such as t1 / 2 alpha, t1 / 2 beta, and area under the curve (AUC). Can do. References include, for example, the experimental part below, as well as standard handbooks (eg Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996)) Made to. A reference is also made in "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The terms “increase in half-life” or “increased half-life” are also as defined in WO 08/020079, page 57, paragraph o), in particular an increase in t1 / 2 beta (t1 / 2alpha and And / or with or without an increase in AUC or both).

  q) With respect to the target or antigen, the term “interaction site” on the target or antigen refers to the site of an amino acid residue on the target or antigen that is the site for binding to a ligand, receptor, or other binding partner; For epitope, antigenic determinant, part, domain, or stretch, catalytic site, cleavage site, site for allosteric interaction, target or antigen multimerization (eg homodimerization or heterodimerization, etc.) Means a site involved; or any other site, epitope, antigenic determinant, moiety, domain, or stretch of amino acid residues on a target or antigen involved in the biological action or mechanism of the target or antigen. More generally, an “interaction site” is a target or antigen (and / or any pathway, interaction, signal that includes a target or antigen) to which an amino acid sequence or polypeptide of the invention can bind. Any site, epitope, antigenic determinant, moiety, amino acid residue on the target or antigen (as defined herein), wherein transmission, biological mechanism, or biological effect) is to be modulated It can be a domain or a stretch.

r) a single variable domain or polypeptide of an immunoglobulin is at least 10 times, such as at least 100 times, greater than the affinity that said amino acid sequence or polypeptide binds to the first antigen to the second target or polypeptide; , preferably at least 1000-fold, and, in as described in 10,000 times or more better affinity / avidity (top, _{K D} value, _{K a value, K off} rate, and / or properly as _{K on} speed Expressed) is said to be “specific” for the first target or antigen as compared to the second target or antigen. For example, the first antigen is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times the KD to which the amino acid sequence or polypeptide binds to the target or antigen to the second target or polypeptide. Binding can be done using a _KD value that is twice as low, eg, 10.000 times less or even less. Preferably, when an immunoglobulin single variable domain or polypeptide is “specific” for a first target or antigen as compared to a second target or antigen, it is directed against said first target or antigen. Directed (as defined herein), but not directed to said second target or antigen.

  s) The terms “cross-block”, “cross-block”, and “cross-block” are used interchangeably herein and a single variable domain or polypeptide of an immunoglobulin is Through the allosteric modulation of a single variable domain or polypeptide of another immunoglobulin of the present invention to its target, meaning the ability to interfere with binding directly or indirectly. The extent to which an immunoglobulin single variable domain or polypeptide of the present invention can interfere with the binding of another to a target, therefore, whether it can be said to cross-block according to the present invention. However, it can be determined using a competitive binding assay. One particular suitable quantitative cross-blocking assay uses a FACS or ELISA-based approach and labels (eg, His-tagged or radioactively labeled) immunoglobulin single variable domains or polypeptides according to the present invention. Competition between the and other binding agents is measured for their binding to the target. The experimental part is generally a suitable FACS for determining whether a binding molecule can cross-block or cross-block an immunoglobulin single variable domain or polypeptide according to the invention, An ELISA, or radioligand displacement based assay is described. It will be appreciated that the assay can be used with any of the immunoglobulin single variable domains or other binding agents described herein. Thus, in general, a cross-blocking amino acid sequence or other binding agent according to the present invention is capable of binding, for example, to a target in the above cross-blocking assay, during the assay, and Test in which in the presence of the second amino acid sequence of the invention or other binding agent, the recorded substitution of an immunoglobulin single variable domain or polypeptide according to the invention is present in an amount of 0.01 mM or less Potential cross-blocking agents (cross-blocking agents can be other conventional monoclonal antibodies such as IgG, classical monovalent antibody fragments (Fab, scFv), etc.) and engineered variants (diabodies, triabodies) , Minibodies, VHH, dAb, VH, VL), the largest theoretical replacement (eg, cold (eg, unlabeled) immunity that needs to be cross-blocked) Between 60% and 100% (eg, in an ELISA / radioligand based competition assay) or between 80% and 100% (eg, in a FACS based competition assay) ).

  t) The amino acid sequence (such as a single variable domain or polypeptide of an immunoglobulin according to the present invention) contains two different antigens or antigenic determinants (eg serum albumin from two different species of mammals, eg human Serum albumin and cynomolgus serum albumin) are said to be “cross-reactive” (if it is specific for both these different antigens or antigenic determinants (as defined herein)).

u) As further described in paragraphs q) of pages 58 and 59 of WO 08/020079 (incorporated herein by reference), the amino acid residues of an immunoglobulin single variable domain may be determined according to Kabat et al. "Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). 23; 240 (1-2): as applied to the V _HH domain from camelids in the literature of 185-195, thus the FR1 of the single variable domain of an immunoglobulin is the amino acid residue at positions 1-30. The CDR1 of the single variable domain of an immunoglobulin contains the amino acid residues at positions 31 to 35, and One variable domain FR2 contains amino acid residues at positions 36-49, an immunoglobulin single variable domain CDR2 contains amino acid residues at positions 50-65, and an immunoglobulin single variable domain FR3 is Wherein the immunoglobulin single variable domain CDR3 comprises amino acid residues at positions 95-102, and the immunoglobulin single variable domain FR4 comprises positions 103-94. Contains 113 amino acid residues.

  v) Figures, sequence listings, and experimental parts / examples are provided only to further illustrate the invention and, unless otherwise clearly indicated herein, the scope of the invention and / or appended claims Should not be construed or understood as limiting in any way.

1. Polypeptides of the invention and uses thereof 1.1. Anti-CXCR7 Building Blocks The polypeptides of the present invention are generally used and CXCR7 and especially human CXCR7 (sequence The binding of No. 1) and in particular inhibit and / or prevent, thus CXCR7 and especially human CXCR7 (SEQ ID No. 1) and / or CXCL12 (and / or CXCL11) and especially human CXCL12 (NM_000609) And / or specifically modulate and specifically inhibit or prevent signal transduction mediated by human CXCL11 (U66096), CXCR7 and especially human CXCR7 (SEQ ID NO: 1) and / or CXC 12 (and / or CXCL11) and in particular human CXCL12 (NM_000609) and / or in particular the biological pathways involving human CXCL11 (U66096) and / or associated with such signaling or these pathways Regulate biological mechanisms, responses, and effects.

  As such, the polypeptides and compositions of the present invention are used for diagnosis, prevention, and treatment of the diseases and disorders of the present invention (herein also “the diseases and disorders of the present invention”). Can, but is not limited to, cancer, such as carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia, adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, Lymphoma, prostate cancer, and Burkitt lymphoma, head and neck cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small intestine cancer, Rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vagina cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine cancer cancer, carcinoid Cancer, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma Non-Hodgkin's lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or AIDS-related primary exudative lymphoma, neuroectodermal tumor, rhabdomyosarcoma (for additional cancers, see Cancer, Principles and practice (DeVita, VT et al. eds 1997)); preferably head and neck cancer, and brain and neurological dysfunction, such as Alzheimer's disease and multiple sclerosis; renal failure, renal allograft rejection; nasal polyps; rheumatoid arthritis; Allograft rejection; cardiac dysfunction; atherosclerosis; asthma; glomerulonephritis; contact dermatitis; inflammatory bowel disease; inflammatory bowel disease; psoriasis; reperfusion injury; Includes other disorders and diseases. In particular, the polypeptides and compositions of the invention are used for diagnosis, prevention, and treatment of diseases including CXCR7-mediated metastasis, chemotaxis, cell adhesion, transendothelial migration, cell proliferation, and / or survival. be able to.

  In general, the “diseases and disorders of the present invention” refer to subjects in need thereof (ie, have the disease or disorder or at least one symptom thereof and / or are at risk of inducing or developing the disease or disorder). And any of the polypeptides or compositions of the invention (and in particular pharmaceutically active amounts thereof) and / or CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) or CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) diagnosis, prevention and / or respectively by appropriate administration of known active ingredients (and in particular their pharmaceutically active amounts) which are active against the biological pathway or mechanism comprising It can be defined as diseases and disorders that can be treated.

  In particular, the polypeptides of the present invention may be produced by excess and / or unwanted CXCL12 and especially human CXCL12 signaling mediated by CXCR7 and especially human CXCR7 (SEQ ID NO: 1), or CXCR7 and especially human CXCR7 (SEQ ID NO: 1). Can be used for diagnosis, prevention, and treatment of the diseases and disorders of the present invention characterized by pathways involved (eg, CXCL11 / I-TAC-CXCR7 axis). Examples of such diseases and disorders of the present invention will again be apparent to those skilled in the art based on the disclosure herein.

  Thus, without being limited thereto, the immunoglobulin single variable domains and polypeptides of the invention are used, for example, CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) mediated signaling (eg Diagnosing, preventing, and / or all diseases and disorders that are currently diagnosed, prevented, or treated using active ingredients that can be modulated (such as those mentioned in the prior art cited in the specification) Can be treated. Also, using the polypeptides of the present invention, all diseases and disorders (for which treatments with such active ingredients are currently being developed, proposed or proposed in the future) It will be envisaged that it can be diagnosed, prevented and / or treated. Also, because of their advantageous properties as further described herein, the polypeptides of the present invention may be other than those in which these known active ingredients are used or proposed or developed. Can be used for the diagnosis, prevention and treatment of other diseases and disorders; and / or the polypeptides of the invention provide new methods and regimens for treating the diseases and disorders described herein. It is assumed that it can be provided.

  Other applications and uses of the immunoglobulin single variable domains and polypeptides of the invention will be apparent to those skilled in the art from the further disclosure herein.

  In general, providing a pharmacologically active agent and a composition comprising the same that can be used in the diagnosis, prevention, and / or treatment of the diseases and / or disorders of the present invention; It is an object of the present invention to provide methods for the diagnosis, prevention and / or treatment of such diseases and disorders, including the administration and / or use of agents and compositions.

  In particular, such pharmacologically active agents, compositions, and methods having certain advantages compared to agents, compositions, and / or methods currently used and / or known in the art. It is an object of the present invention to provide a method. These advantages will become clear from the further description below.

  More particularly, pharmacologically active agents for the diagnosis, prevention and / or treatment of the diseases and / or disorders of the present invention and of the further diseases and disorders referred to herein, and compositions comprising the same For the diagnosis, prevention, and / or treatment of such diseases and disorders, including the administration and / or use of such therapeutic proteins and compositions It is an object of the present invention to provide this method.

  Accordingly, immunoglobulins directed against CXCR7, in particular against CXCR7 from warm-blooded animals, more particularly against CXCR7 from mammals (such as mice), in particular against human CXCR7 (SEQ ID NO: 1) It is a specific object of the present invention to provide a single variable domain of the present invention; and to provide proteins and polypeptides comprising or consisting essentially of at least one such immunoglobulin single variable domain .

  In particular, such immunoglobulin single variable domains and such proteins and / or suitable for prophylactic, therapeutic and / or diagnostic use in warm-blooded animals, especially mammals, and more particularly humans It is a specific object of the present invention to provide a polypeptide.

  More particularly, prevention, treatment, alleviation of one or more diseases, disorders, or conditions associated with and / or mediated by CXCR7, such as the diseases, disorders, and conditions referred to herein. And / or such immunoglobulin single variable domains and such proteins and / or polypeptides that can be used in warm-blooded animals, in particular in mammals, and more particularly in humans, for diagnosis. This is a specific object of the present invention.

  Also, for the prevention and / or treatment of one or more diseases, disorders, or conditions associated with and / or mediated by CXCR7 (eg, diseases, disorders, and conditions referred to herein), Such immunoglobulin single variable domains and such proteins and / or that can be used in preparations of pharmaceutical or veterinary compositions in warm-blooded animals, particularly in mammals, and more particularly in humans It is a specific object of the present invention to provide a polypeptide.

  In the present invention, these objects are generally achieved through the use of immunoglobulin single variable domains, proteins, polypeptides, and compositions as described herein.

  In general, the present invention is directed against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) (as defined herein) and / or can specifically bind thereto (as defined herein). As provided) single variable domains of immunoglobulins; and compounds and constructs, and in particular proteins and polypeptides, comprising at least one such amino acid sequence.

More particularly, the present invention is to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) are as defined herein affinity _{(K D} values (on an actual or apparent), _{K A} value (actual or Immunity that can be bound using (appearance), K _on rate and / or K _off rate, or as measured and / or represented appropriately as IC ₅₀ values and as further described herein) Globulin single variable domains and polypeptides; and compounds and constructs, and in particular, proteins and polypeptides, comprising at least one such amino acid sequence.

In certain aspects, the immunoglobulin single variable domains and / or polypeptides of the invention are:
-Human CXCR7 (SEQ ID NO: 1) with an EC50 of less than 100 nM, more preferably less than 50 nM, even more preferably less than 20 nM, most preferably less than 10 nM, in a binding FACS assay, eg Binds as described in Example 8), wherein the polypeptide comprises only one single variable domain unit of human CXCR7 binding immunoglobulin;
And / or they are:
-Human CXCL12 (SDF-1) from human CXCR7 (SEQ ID NO: 1) with an average Ki value of 100 nM or less, more preferably with an average Ki value of 20 nM or less, even more preferably with an average Ki value of 10 nM or less, In the assay, for example, as described in the experimental part (Examples 9 and 10), complete substitutions, wherein the polypeptide comprises only one single variable domain unit of human CXCR7 binding immunoglobulin, Here, complete substitution means an average CXCL12 substitution of about 60% to 80% and higher (as measured according to the ligand displacement assay of Example 9), or where complete substitution is about 80% to Means average CXCL12 substitution of 100% and above (as measured according to the FACS-based competition assay of Example 10);
And / or they are:
-Human CXCL11 (I-TAC) from human CXCR7 (SEQ ID NO: 1) with an average Ki value of 1000 nM or less, more preferably an average Ki value of 500 nM or less, even more preferably an average Ki value of 100 nM or less, even more Preferably with an average Ki value of 20 nM or less, even more preferably an average Ki value of 10 nM or less, in the assay, for example as described in the experimental part (Examples 9 and 10), where The polypeptide contains only one single variable domain unit of human CXCR7 binding immunoglobulin, where complete substitution means about 60% to 80% and more average CXCL11 substitution (of Example 9). Or as determined here according to the ligand displacement assay), or where complete substitution is an average CXCL12 substitution of about 80% to 100% and above. Taste and (when measured according to FACS-based competition assay of Example 10);
And / or they are:
-Human CXCL12 (SDF-1) from human CXCR7 (SEQ ID NO: 1) with an average Ki value of 100 nM or less, more preferably with an average Ki value of 20 nM or less, even more preferably with an average Ki value of 10 nM or less, In the assay, for example, partially substituted as described in the experimental part (Examples 9 and 10), where the polypeptide contains only one single variable domain unit of human CXCR7 binding immunoglobulin. Where partial substitution means about 40% to 60% average CXCL12 substitution (as measured according to the ligand displacement assay of Example 9) or where partial substitution is about 50% to Means an average CXCL12 displacement of 80% (when measured according to the FACS-based competition assay of Example 10);
And / or they are:
-Human CXCL11 (I-TAC) from human CXCR7 (SEQ ID NO: 1) with an average Ki value of 1000 nM or less, more preferably an average Ki value of 500 nM or less, even more preferably an average Ki value of 100 nM or less, even more Preferably, with an average Ki value of 20 nM or less, even more preferably an average Ki value of 10 nM or less, in the assay, for example as described in the experimental part (Examples 9 and 10), where a partial replacement , The polypeptide comprises only one single variable domain unit of human CXCR7 binding immunoglobulin, where partial substitution means an average CXCL11 substitution of about 40% to 60% (ligand of Example 9). Or, where partial substitution means about 50% to 80% average CXCL12 substitution (as measured according to the displacement assay) (of Example 10). When measured in accordance with ACS-based competition assay);
And / or they are:
Human CXCR7 (SEQ ID NO: 1) has an average Kd value of 100 nM or less, more preferably an average Kd value of 50 nM or less, even more preferably 40 nM or less, eg less than 30, 25, 20, 15, 10, 9 , 8, 7, 6, 5, 4, 3, 3 nM or less, such as less than 1 nM, or most preferably still with an average Kd value of less than 0.1 nM.

  Binding of a single variable domain and / or polypeptide of an immunoglobulin of the invention to (human) CXCR7 may involve substitution of (human) CXCL11 and / or CXCL12 from (human) CXCR7, as described herein. It should be recognized that this can be done. Binding of a single variable domain and / or polypeptide of an immunoglobulin of the present invention to (human) CXCR7, as described herein, may include (human) CXCR11 to its cognate receptor, such as (human) CXCR7. It should further be appreciated that and / or can result in inhibition of CXCR12 binding.

As already mentioned, in some specific but non-limiting aspects (described in more detail herein), the present invention provides:
Directed to CXCR7 (as defined herein) and inhibits or blocks (fully or partially as further described herein) ligand binding, and in particular to CXCR7 An amino acid sequence capable of inhibiting or blocking (fully or partially as further described herein) binding of SDF-1 (as described further herein). These amino acid sequences are also referred to herein as “CXCR-7 binding amino acid sequences” or “CXCR7 binding blocks”. Preferably, these CXCR7 binding amino acid sequences are ISVD (as described herein), in which case they are also referred to as “CXCR7 binding ISVD”. Preferably, any CXCR7 binding amino acid sequence, CXCR7 binding building block, or CXCR7 binding ISVD has a blocking activity, ie, partially or completely blocks SDF-1 binding to CXCR7; It can be determined by any suitable assay known to those skilled in the art, for example, by Alphascreen assay or by FACS competition assay (eg, as described herein). Preferably, blocking activity is determined by FACS competition assay as described in Example 9. Preferably, the ISVD has blocking activity or competitive ability of CXCR7 or competitive SDF-1 binding in NIH3T3-hCXCR7 cells and is less than 600 nM, but preferably 500 nM, 400 nM, 300 nM, 200 nM, 100 nM or In addition, it has a lower average Ki.

  -For example, 01C10-like ISVD has blocking activity or competitive ability in this assay and is less than 100 nM, more preferably less than 75 nM, less than 50 nM, or even less, such as less than 40 nM or 30 nM, 25 nM or 24 nM, or even more More preferably with an average Ki of less than 22 nM.

  -For example, 14G03-like ISVD has blocking activity or competitive ability in this assay and is less than 150 nM, more preferably less than 100 nM, 90 nM, less than 80 nM, or even lower, such as 70 nM or 60 nM, 50 nM or 40 nM, 30 nM, With an average Ki of 20 nM, 15 nM or 10 nM, 5 nM, or even more preferably less than 4 nM.

  In one specific but non-limiting aspect, a “CXCR-7 binding amino acid sequence” or “CXCR7 binding block” (part of) is capable of inhibiting or blocking β-arrestin mobilization. It can be (and preferably also is). Preferably, any CXCR7 binding amino acid sequence, CXCR7 binding building block, or CXCR7 binding ISVD allows them to have blocking activity (ie, partially or completely block SDF-1-mediated CXCR7 signaling) It can be determined by any suitable assay known to those of skill in the art (eg, by any suitable β-arrestin mobilization assay) as described herein.

  Preferably, blocking activity or inhibitory capacity is determined by β-arrestin assay as described in Example 15. Preferably, the ISVD has a ligand (eg, SDF-1) -induced β-arrestin blocking activity or inhibitory ability in the PathHunter eXpress β-arrestin assay (DiscoverX), greater than 25%, greater than 30%, but preferably Is associated with% inhibition of β-arrestin mobilization by 40%, 50%, 60%, 70%, 80% or even more.

  -For example, 14G03-like ISVD has blocking activity or inhibitory capacity in this assay and is greater than 50%, more preferably greater than 60%, 70% or even greater, such as greater than 75% or 80%, etc. 85% or even more, preferably with a% inhibition of more than 90%.

  Some preferred techniques for binding, substitution, migration, or other in vivo and / or in vitro efficacy of a single variable domain or polypeptide of an immunoglobulin of the invention to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) Target values will become clear from the further description and examples herein.

  In the description and claims, the following terms are defined as follows:

A) 01C10-like sequence: A “01C10-like sequence”, “01C10-like ISVD”, “01C10-like building block” or “Group 1 ISVD” is defined as an ISVD (as described herein) comprising:
a) (i) the amino acid sequence NYAMG (SEQ ID NO: 93) or (ii) an amino acid sequence having only 3, 2, or 1 amino acid difference (as defined herein) from the amino acid sequence NYAMG (SEQ ID NO: 93) CDR1 comprising or consisting essentially of either; and / or b) (i) amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95) or (ii) amino acid sequence AITPRAFTYTYADSVKG (SEQ ID NO: 95) and at least 80%, such as at least 85%, For example, an amino acid sequence having at least 90%, or greater than 95% sequence identity; or (iii) Only 7, 6, 5, 4, 3, 2, or 1 amino acid difference from amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95) (As defined in this specification And / or c) (i) the amino acid sequence QLVGSSGNLGRQESYAY (SEQ ID NO: 97) or (ii) the amino acid sequence QLVGSSNLGGRQUESAY (SEQ ID NO: 97) and at least 80%, such as at least 85%, such as An amino acid sequence having at least 90%, or greater than 95% sequence identity; or (iii) a difference of only 7, 6, 5, 4, 3, 2, or 1 amino acid from amino acid sequence QLVGSGSNLGLQUESAY (SEQ ID NO: 97) CDR3 which is any of the amino acid sequences having (as defined herein);
Here, the framework sequences present in such ISVD are as further described herein, where CDR1, CDR2, and CDR3 preferably have a 01C10-like ISVD having blocking activity, For example, partially or completely blocking CXCL11 and / or CXCL12 binding to CXCR7 (as described above) and / or reducing and / or inhibiting tumor formation in a xenograft model and / or CXCR7 It binds to and / or recognizes amino acid residue W19 in (SEQ ID NO: 1), and optionally amino acid residue S23 and / or amino acid residue D25 (all as described herein).

  Also, as mentioned herein, 01C10-like sequences (parts) are displacement assays where they use SDF-1 binding (see Examples 9 and 10), eg, in Example 10. Can be inhibited (blocked) or replaced (and preferably also). Preferably, in such 01C10-like sequences, CDR1 and CDR2 are as defined under a) and b), respectively; or CDR1 and CDR3 are defined under a) and c), respectively. Or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such 01C10-like sequences, CDR1, CDR2, and CDR3 are all as defined under a), b), and c), respectively. Again, in such 01C10-like sequences, CDR1, CDR2, and CDR3 preferably have partial or complete blocking of SDF-1 binding to CXCR7, for example, 01C10-like ISVD has blocking activity (this And / or reduces and / or inhibits tumor formation in a xenograft model and / or amino acid residue W19 in CXCR7 (SEQ ID NO: 1), and optionally an amino acid residue, as described in the specification) Binds to and / or recognizes S23 and / or amino acid residue D25 (all as described herein).

For example, in such a 01C10-like sequence: CDR1 may comprise or substantially consist of the amino acid sequence NYAMG (SEQ ID NO: 93) (with CDR2 and CDR3 as defined below under b) and c), respectively) And / or CDR2 can comprise or consist essentially of the amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95) (with CDR1 and CDR3 as defined under a) and c), respectively) And / or CDR3 may comprise or consist essentially of the amino acid sequence QLVGSGSNLGLQESYAY (SEQ ID NO: 97) (with CDR1 and CDR2 as defined under a) and b), respectively). In particular, if a 01C10-like sequence follows this aspect: CDR1 can comprise or consist essentially of the amino acid sequence NYAMG (SEQ ID NO: 93), and CDR2 can comprise the amino acid sequence AITPRAFTYTYADSVKG (SEQ ID NO: 95) (c above With or without CDR3 as defined below) and / or CDR1 can comprise or consist essentially of the amino acid sequence NYAMG (SEQ ID NO: 93) And CDR3 can comprise or consist essentially of the amino acid sequence QLVGSSGNLGRQESYAY (SEQ ID NO: 97) (with CDR2 as defined under (b) above); and / or CDR2 , Amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95 And the CDR3 can comprise or substantially comprise the amino acid sequence QLVGSSGSNLGRQESYAY (SEQ ID NO: 97) (with CDR1 as defined under a) above) Can be. Again, in such 01C10-like sequences, CDR1, CDR2, and CDR3 preferably have partial or complete blocking of SDF-1 binding to CXCR7, for example, 01C10-like ISVD has blocking activity (this And / or reduces and / or inhibits tumor formation in a xenograft model and / or amino acid residue W19 in CXCR7 (SEQ ID NO: 1), and optionally an amino acid residue, as described in the specification) Binds to and / or recognizes S23 and / or amino acid residue D25 (all as described herein). In a particularly preferred aspect, the “01C10-like sequence”, “01C10-like ISVD”, “01C10-like building block”, or “Group 1 ISVD” is an ISVD comprising:
d) (i) the amino acid sequence NYAMG (SEQ ID NO: 93) or (ii) an amino acid sequence having only 3, 2, or 1 amino acid difference (as defined herein) from the amino acid sequence NYAMG (SEQ ID NO: 93). And / or e) (i) the amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95) or (ii) the amino acid sequence AITPRAFTYTYADSVVG (SEQ ID NO: 95) and at least 80%, such as at least 85%, such as at least 90%, Or an amino acid sequence having greater than 95% sequence identity; or (iii) Amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95) and only 7, 6, 5, 4, 3, 2, or 1 amino acid difference (as defined herein) Of amino acid sequence having And / or f) (i) amino acid sequence QLVGSSNSLGRQESYAY (SEQ ID NO: 97) or (ii) amino acid sequence QLVGSGSNLGLQESYAY (SEQ ID NO: 97) and at least 80%, such as at least 85%, such as at least 90%, Or an amino acid sequence having greater than 95% sequence identity; or (iii) a difference of only 7, 6, 5, 4, 3, 2, or 1 amino acid from the amino acid sequence QLVGSGSNLGLQESYAY (SEQ ID NO: 97) as defined herein CDR3, which is any of the amino acid sequences having the same; wherein the framework sequences present in such ISVD are as further described herein, where CDR1, CDR2, and CDR3 Is preferably 01C1 Like ISVD has blocking activity, eg, partially or completely blocks SDF-1 binding to CXCR7 (as described herein) and / or reduces tumor formation and / or in xenograft models Or inhibit and / or bind to and / or recognize amino acid residue W19 in CXCR7 (SEQ ID NO: 1) and optionally amino acid residue S23 and / or amino acid residue D25 (all As described in the description). Preferably, in a 01C10-like sequence according to this specifically preferred aspect, CDR1 and CDR2 are defined under d) and e), respectively; or CDR1 and CDR3 are respectively d) and f) Or CDR2 and CDR3 are defined under e) and f), respectively. More preferably, in such 01C10-like sequences, CDR1, CDR2, and CDR3 are all defined under d), e), and f), respectively. Again, in such 01C10-like sequences, CDR1, CDR2, and CDR3 preferably have partial or complete blocking of SDF-1 binding to CXCR7, for example, 01C10-like ISVD has blocking activity (this And / or reduces and / or inhibits tumor formation in a xenograft model and / or amino acid residue W19 in CXCR7 (SEQ ID NO: 1), and optionally an amino acid residue, as described in the specification) Binds to and / or recognizes S23 and / or amino acid residue D25 (all as described herein).

  For example, in a 01C10-like sequence according to this specifically preferred aspect: with CDR1 and CDR3 as defined below under the amino acid sequence NYAMG (SEQ ID NO: 93) (e and f respectively) And / or CDR2 is the amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95) (with CDR1 and CDR3 as defined below respectively); and / or CDR3 is an amino acid The sequence QLVGSSGNLGRQESYAY (SEQ ID NO: 97) (with CDR1 and CDR2 as defined below under d) and e)). In particular, if a 01C10-like sequence follows this aspect: CDR1 is the amino acid sequence NYAMG (SEQ ID NO: 93) and CDR2 is defined under the amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95) (f above) And / or CDR1 is the amino acid sequence NYAMG (SEQ ID NO: 93) and CDR3 is under the amino acid sequence QLVGSGSNLLGQESYAY (SEQ ID NO: 97) (e above) And / or CDR2 is the amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95) and CDR3 is the amino acid sequence QLVGSGSNLGLQESYAY (SEQ ID NO: 97) (above d) As defined below That CDR1 is the accompanying). Again, in such 01C10-like sequences, CDR1, CDR2, and CDR3 preferably have partial or complete blocking of SDF-1 binding to CXCR7, for example, 01C10-like ISVD has blocking activity (this And / or reduces and / or inhibits tumor formation in a xenograft model and / or amino acid residue W19 in CXCR7 (SEQ ID NO: 1), and optionally an amino acid residue, as described in the specification) Binds to and / or recognizes S23 and / or amino acid residue D25 (all as described herein).

  A particularly preferred 01C10-like sequence: CDR1 is the amino acid sequence NYAMG (SEQ ID NO: 93), CDR2 is the amino acid sequence AITPRAFTYTYYADSVKG (SEQ ID NO: 95); and CDR3 is the amino acid sequence QLVGSGSNLGLQUESTAY (SEQ ID NO: 97).

  In all 01C10-like sequences described in this paragraph A), framework sequences can be further described herein. Preferably, the framework sequence has at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity with the framework sequence of 01C10 (it is Can be determined, for example, by determining the overall degree of sequence identity between the sequence of 01C10 and a given sequence, ignoring CDRs in the calculation). Again, the combination of CDRs and frameworks present in a given sequence preferably results in partial or complete blockage of SDF-1 binding to, for example, CXCR7, where the resulting 01C10-like ISVD has blocking activity. And / or reduce and / or inhibit tumor formation in a xenograft model and / or amino acid residue W19 in CXCR7 (SEQ ID NO: 1), and optionally an amino acid Bind to and / or recognize residue S23 and / or amino acid residue D25 (all as described herein).

  In one particular aspect, the 01C10-like sequence is at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, or more than 95% sequence identical to the amino acid sequence 01C10 (SEQ ID NO: 91) ISVD having the property. For example, in a 01C10-like sequence according to this aspect, the CDRs can follow the specifically preferred aspects described above, but in particular (but without limitation) NYAMG (SEQ ID NO: 93) (CDR1); AITPRAFTTYYADSVKG ( SEQ ID NO: 95) (CDR2); and QLVGSSGNLGRQUESAY (SEQ ID NO: 97) (CDR3). Again, preferably, the combination of CDRs and frameworks present in such 01C10-like ISVD preferably has a resulting 01C10-like ISVD having blocking activity, eg, partially binding SDF-1 to CXCR7. And / or reduce and / or inhibit tumor formation in a xenograft model and / or amino acid residue W19 in CXCR7 (SEQ ID NO: 1), and / or completely block (as described herein), and / or , Optionally binds to and / or recognizes amino acid residue S23 and / or amino acid residue D25 (all as described herein). In one particular aspect, any 01C10-like sequence can be humanized and / or sequence optimized as further described herein.

B) 14G03-like sequence: A “14G03-like sequence”, “14G03-like ISVD”, “14G03-like building block” or “Group 2 ISVD” is defined as an ISVD (as described herein) comprising:
a) (i) the amino acid sequence INYMG (SEQ ID NO: 13) or (ii) an amino acid sequence having only 3, 2, or 1 amino acid difference (as defined herein) from the amino acid sequence INYMG (SEQ ID NO: 13) CDR1 comprising or consisting essentially of either; and / or b) (i) amino acid sequence TLTSGGSTNYAGSVKG (SEQ ID NO: 23) or (ii) amino acid sequence TLTSGGSTNYAGSVVG (SEQ ID NO: 23) and at least 80%, such as at least 85%, For example, an amino acid sequence having at least 90%, or greater than 95% sequence identity; or (iii) Only 7, 6, 5, 4, 3, 2, or 1 amino acid difference from amino acid sequence TLTSGGGSTNYAGSVKG (SEQ ID NO: 23) (As defined in this specification) And / or c) (i) the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) or (ii) the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33), at least 80%, such as at least 85%, for example, An amino acid sequence having at least 90% or greater than 95% sequence identity; or (iii) a difference of only 7, 6, 5, 4, 3, 2, or 1 amino acid from the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) (this CDR3 which is any of the amino acid sequences having (as defined in the specification);
Here, the framework sequences present in such ISVD are as further described herein, where CDR1, CDR2, and CDR3 preferably have 14G03-like ISVDs having blocking activity, For example, partially or completely blocking CXCL11 and / or CXCL12 binding to CXCR7 (as described above) and / or reducing and / or inhibiting tumor formation in a xenograft model and / or β Inhibits arrestin mobilization and / or binds to and / or recognizes amino acid residue M33 in CXCR7 (SEQ ID NO: 1) and, optionally, amino acid residue V32 and / or amino acid residue M37 (all , As described herein).

  Also, as referred to herein, 14G03-like sequences (a portion of) are displacement assays where they use SDF-1 binding (see Examples 9 and 10), eg, in Example 10. Can be inhibited (blocked) or replaced (and preferably also). Preferably, in such a 14G03-like sequence, CDR1 and CDR2 are as defined under a) and b) respectively; or CDR1 and CDR3 are defined under a) and c), respectively. Or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 14G03-like sequence, CDR1, CDR2, and CDR3 are all as defined under a), b), and c), respectively. Again, in such 14G03-like sequences, CDR1, CDR2, and CDR3 preferably have partial or complete blocking of SDF-1 binding to CXCR7, for example, 14G03-like ISVD has blocking activity (this And / or reduces and / or inhibits tumor formation in a xenograft model and / or amino acid residue M33 in CXCR7 (SEQ ID NO: 1), and optionally amino acid residues, as described in the specification) It binds to and / or recognizes V32 and / or amino acid residue M37 (all as described herein).

  For example, in such a 14G03-like sequence: CDR1 may comprise or substantially comprise the amino acid sequence INYMG (SEQ ID NO: 13) (with CDR2 and CDR3 as defined below under b) and c), respectively) And / or CDR2 can comprise or consist essentially of the amino acid sequence TLTSGGSTNYAGSVVG (SEQ ID NO: 23) (with CDR1 and CDR3 as defined under a) and c), respectively) And / or CDR3 can comprise or consist essentially of the amino acid sequence GGTLYDRRRFES (SEQ ID NO: 33) (with CDR1 and CDR2 as defined below under a) and b), respectively. In particular, if a 14G03-like sequence follows this aspect: CDR1 can comprise or consist essentially of the amino acid sequence INYMG (SEQ ID NO: 13), and CDR2 can comprise the amino acid sequence TLTSGGSTNYAGSVVG (SEQ ID NO: 23) (c above With or without CDR3 as defined below); and / or CDR1 can comprise or consist essentially of the amino acid sequence INYMG (SEQ ID NO: 13). And CDR3 can comprise or consist essentially of the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) (with CDR2 as defined under (b) above; and / or CDR2 , Which may comprise the amino acid sequence TLTSGGSTNYAGSVKG (SEQ ID NO: 23) Then may become substantially and, CDR3 comprises the amino acid sequence GGTLYDRRRFES (SEQ ID NO: 33) can be a substantially CDR1 are as defined accompanied) includes can or from it under the (a above). Again, in such 14G03-like sequences, CDR1, CDR2, and CDR3 preferably have partial or complete blocking of SDF-1 binding to CXCR7, for example, 14G03-like ISVD has blocking activity (this And / or reduces and / or inhibits tumor formation in a xenograft model and / or amino acid residue M33 in CXCR7 (SEQ ID NO: 1), and optionally amino acid residues, as described in the specification) It binds to and / or recognizes V32 and / or amino acid residue M37 (all as described herein).

In a particularly preferred aspect, the “14G03-like sequence”, “14G03-like ISVD”, “14G03-like building block”, or “Group 2 ISVD” is an ISVD comprising:
d) (i) the amino acid sequence INYMG (SEQ ID NO: 13) or (ii) the amino acid sequence INYMG (SEQ ID NO: 13) and only 3, 2, or 1 amino acid difference (as defined herein) And / or e) (i) amino acid sequence TLTSGGSTNYAGSVKG (SEQ ID NO: 23) or (ii) amino acid sequence TLTSGGSTNYAGSVVG (SEQ ID NO: 23) and at least 80%, such as at least 85%, such as at least 90% or An amino acid sequence having greater than 95% sequence identity; or (iii) a difference of only 7, 6, 5, 4, 3, 2, or 1 amino acid from the amino acid sequence TLTSGGSTNYAGSVKG (SEQ ID NO: 23) (as defined herein) Any of the amino acid sequences having And / or f) (i) the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) or (ii) the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) and at least 80%, such as at least 85%, such as at least 90%, or 95%. An amino acid sequence having a sequence identity greater than; or (iii) Amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) and only 7, 6, 5, 4, 3, 2, or 1 amino acid difference (as defined herein) CDR3 which is any of the amino acid sequences having
Here, the framework sequences present in such ISVD are as further described herein, where CDR1, CDR2, and CDR3 preferably have 14G03-like ISVDs having blocking activity, For example, partially or completely blocking SDF-1 binding to CXCR7 (as described herein) and / or reducing and / or inhibiting tumor formation in a xenograft model and / or β Inhibits arrestin mobilization and / or binds to and / or recognizes amino acid residue M33 in CXCR7 (SEQ ID NO: 1) and, optionally, amino acid residue V32 and / or amino acid residue M37 (all , As described herein). Preferably, in the 14G03-like sequence according to this specifically preferred aspect, CDR1 and CDR2 are as defined under d) and e), respectively; or CDR1 and CDR3 are d) and f, respectively. ); Or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 14G03-like sequence, CDR1, CDR2, and CDR3 are all as defined under d), e), and f), respectively. Again, in such 14G03-like sequences, CDR1, CDR2, and CDR3 preferably have partial or complete blocking of SDF-1 binding to CXCR7, for example, 14G03-like ISVD has blocking activity (this And / or reduces and / or inhibits tumor formation in a xenograft model and / or amino acid residue M33 in CXCR7 (SEQ ID NO: 1), and optionally amino acid residues, as described in the specification) It binds to and / or recognizes V32 and / or amino acid residue M37 (all as described herein).

  For example, in a 14G03-like sequence according to this specifically preferred aspect: with CDR1 and CDR2 and CDR3 as defined under the amino acid sequence INYMG (SEQ ID NO: 13) (e and f respectively) And / or CDR2 is the amino acid sequence TLTSGGSTNYAGSVKG (SEQ ID NO: 23) (with CDR1 and CDR3 as defined below respectively); and / or CDR3 is an amino acid The sequence GGTLYDRRRFES (SEQ ID NO: 33) (with CDR1 and CDR2 as defined under d) and e), respectively). In particular, if a 14G03-like sequence follows this aspect: CDR1 is the amino acid sequence INYMG (SEQ ID NO: 13) and CDR2 is defined under the amino acid sequence TLTSGGSTNYAGSVVG (SEQ ID NO: 23) (above f) And / or CDR1 is the amino acid sequence INYMG (SEQ ID NO: 13) and CDR3 is under the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) (e above) And / or CDR2 is the amino acid sequence TLTSGGSTNYAGSVKG (SEQ ID NO: 23) and CDR3 is the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) (d above) With CDR1 as defined below) . Again, in such 14G03-like sequences, CDR1, CDR2, and CDR3 preferably have partial or complete blocking of SDF-1 binding to CXCR7, for example, 14G03-like ISVD has blocking activity (this And / or reduces and / or inhibits tumor formation in a xenograft model and / or amino acid residue M33 in CXCR7 (SEQ ID NO: 1), and optionally amino acid residues, as described in the specification) It binds to and / or recognizes V32 and / or amino acid residue M37 (all as described herein).

  In a particularly preferred 14G03-like sequence: CDR1 is the amino acid sequence INYMG (SEQ ID NO: 13), CDR2 is the amino acid sequence TLTSGGSTNYAGSVKG (SEQ ID NO: 23); and CDR3 is the amino acid sequence GGTLYDRRRRFES (SEQ ID NO: 33) .

  In all 14G03-like sequences described in this paragraph A), the framework sequences can be as further described herein. Preferably, the framework sequence has at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity with the framework sequence of 14G03 ( It can be determined, for example, by determining the overall degree of sequence identity between a given sequence and the sequence of 14G03, with CDRs ignored in the calculation). Again, the combination of CDR and framework present in a given sequence preferably results in partial or complete blocking of SDF-1 binding to, for example, CXCR7, where the resulting 14G03-like ISVD has blocking activity. And / or reduce and / or inhibit tumor formation in a xenograft model and / or inhibit β-arrestin mobilization and / or in CXCR7 (SEQ ID NO: 1) It binds to and / or recognizes amino acid residue M33, and optionally amino acid residue V32 and / or amino acid residue M37 (all as described herein).

  In one particular aspect, the 14G03-like sequence has at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, or 95% sequence identity with the amino acid sequence 14G03 (SEQ ID NO: 43). ISVD having For example, in a 14G03-like sequence according to this aspect, the CDR may follow the specifically preferred aspects described above, in particular (but without limitation) INNYMG (SEQ ID NO: 13) (CDR1); TLTSGGSTNYAGSVKG (sequence No. 23) (CDR2); and GGTLYDRRRFES (SEQ ID NO: 33) (CDR3). Again, preferably, the combination of CDRs and frameworks present in such 14G03-like ISVD preferably has a resultant 14G03-like ISVD having blocking activity, eg, partially binding SDF-1 to CXCR7. And / or block (as described herein) and / or reduce and / or inhibit tumor formation in a xenograft model and / or inhibit β-arrestin mobilization and / or CXCR7 (sequence Bind to and / or recognize amino acid residue M33 and optionally amino acid residue V32 and / or amino acid residue M37 in number 1) (all as described herein) . In one particular aspect, any 14G03-like sequence can be humanized and / or sequence optimized as further described herein.

  For binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), the amino acid sequence or polypeptide of the invention will usually have one or more amino acid residues or one of the amino acid residues within the amino acid sequence. Or a plurality of stretches (i.e., adjacent to each other or in close proximity to each other, i.e., with each "stret" comprising two or amino acid residues in the primary or tertiary structure of the amino acid sequence), Through it, the amino acid sequence of the present invention can bind to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), the amino acid residue or stretch of amino acid residues is thus CXCR7 and in particular human CXCR7. Forms a “site” for binding to (SEQ ID NO: 1) (also referred to herein as “antigen binding site”) .

  An immunoglobulin single variable domain provided by the present invention is preferably in a substantially isolated form (as defined herein) or comprises a portion of a protein or polypeptide of the invention. Formed (as defined herein), which may comprise or consist essentially of a single variable domain of one or more immunoglobulins of the invention, and optionally may comprise one or A plurality of additional immunoglobulin single variable domains (all optionally linked via one or more suitable linkers) and / or one or more additional binding domains, binding units, amino acid sequences , Or other (functional) groups or moieties, but they preferably also confer one or more desired properties to the construct Non-limiting examples of some dough will become apparent from the further description herein).

  The polypeptide or immunoglobulin variable domains provided by the present invention preferentially reduce tumor formation in vivo.

  In a further preferred embodiment, the present invention provides a construct comprising at least two immunoglobulin single variable domains for CXCR7. More preferably, the immunoglobulin single variable domain for CXCR7 is selected from a polypeptide for CXCR7 and a variant of the immunoglobulin single variable domain, as defined in Section 1.5 for Table B-2 below. (Eg, a single variable domain of an immunoglobulin of group 2), wherein the single variable domain of said immunoglobulin for CXCR7 can be the same or different. Preferably, the two immunoglobulin single variable domains for CXCR7 are selected from 14G03-like ISVDs (eg 14G03, 08A05, 08A10, 07C03, and 07B11, etc.). In another more preferred embodiment, the present invention provides a construct comprising at least two immunoglobulin single variable domains for CXCR7, as shown in Table B-2 below (eg, Group 1 immunoglobulin single variable domains). ), As defined in section 1.5, is selected from polypeptides to CXCR7 and immunoglobulin single variable domain variants, wherein the immunoglobulin single variable domains for CXCR7 may be the same or different. Preferably, said two immunoglobulin single variable domains for CXCR7 are selected from 01C10 (SEQ ID NO: 91), 01B12 (SEQ ID NO: 100), 01F11 (SEQ ID NO: 101), or 01B10 (SEQ ID NO: 102).

  It was unexpectedly demonstrated that a bispecific construct comprising at least one group 1 immunoglobulin single variable domain and at least one group 2 ISVD is particularly suitable for reducing tumor growth in vivo. ing. In particular, these constructs have been shown to inhibit SDF-1 binding to CXCR7, inhibit tumor growth in vivo, and inhibit β-arrestin mobilization. Furthermore, in terms of the binding efficacy of group 2 ISDV, these constructs comprising at least one group 1 ISVD and at least one group 2 ISVD may be Bind well (eg, see Example 17). This can result in a lower dose to inhibit tumor growth. Also, simultaneous inhibition of β-arrestin mobilization can lead to long-term antitumor effects.

  Accordingly, in a further preferred embodiment, the present invention provides a construct comprising at least two immunoglobulin single variable domains for CXCR7, wherein at least one of said immunoglobulin single variable domains for CXCR7 ( That is, the “first” immunoglobulin single variable domain for CXCR7 is 01C10-like, eg, 01C10 (SEQ ID NO: 91), 01B12 (SEQ ID NO: 100), 01F11 (SEQ ID NO: 101), or 01B10 (SEQ ID NO: 102). Or variants thereof as defined in Section 1.5 below for Table B-2 (eg, a single variable domain of a Group 1 immunoglobulin), wherein at least one immunoglobulin single molecule to CXCR7. One variable domain That is, the “secondary immunoglobulin single variable domain for CXCR7) is different from the“ first ”immunoglobulin single variable domain for CXCR7 or variants thereof in Section 1.5 for Table B-2 below. It is selected from polypeptides as defined and variants of immunoglobulin single variable domains. Preferably, the single variable domain of the “first” immunoglobulin for CXCR7 is 01C10, and the single variable domain of the “second” immunoglobulin for CXCR7 is 14G03-like, eg 14G03, 08A05, 08A10, 07C03, And 07B11.

  As described in Example 11, binding to CXCR7 by a single variable domain of a group 1 immunoglobulin, as represented by 01C10, is affected by mutating W19. In contrast, single tested variable domain binding of all tested immunoglobulins was affected by the M33 mutation, but not the Group 1 ISVD. It was further shown that Group 1 ISVDs preferably recognize and / or bind to S23 and D25 (data not shown).

  Group 1 ISVDs or polypeptides can be characterized by binding / recognizing a “Group 1 epitope”. Group 1 ISVDs or polypeptides bind to and / or recognize amino acid residue W19 in CXCR7 (SEQ ID NO: 1), and optionally amino acid residue S23 and / or amino acid residue D25. Group 1 epitopes include amino acid residue W19 in CXCR7 (SEQ ID NO: 1), and optionally amino acid residue S23 and / or amino acid residue D25. Group 1 ISVDs are represented, inter alia, by 01C10 (SEQ ID NO: 91), 01B12 (SEQ ID NO: 100), 01F11 (SEQ ID NO: 101), or 01B10 (SEQ ID NO: 102), clearly distinct from the epitope of group 2 Hit an epitope of;

  Group 2 ISVDs or polypeptides can be characterized by binding / recognizing a “Group 2 epitope”. Group 2 ISVDs or polypeptides do not bind to and / or do not recognize amino acid residue W19, amino acid residue S23, and / or amino acid residue D25 in CXCR7 (SEQ ID NO: 1). Group 2 ISVDs or polypeptides bind to and / or recognize amino acid residue M33 and optionally amino acid residue V32 and / or amino acid residue M37 in CXCR7 (SEQ ID NO: 1). Group 2 epitopes comprise amino acid residue M33 and optionally amino acid residue V32 and / or amino acid residue M37 in CXCR7 (SEQ ID NO: 1). Group 2 ISVDs are represented by 14G03-like ISVDs, such as 14G03 (09A04), 08A05, 08A10, and 07C03, clearly hitting an epitope distinct from Group 1. Preferably, the Group 2 ISVD inhibits β-arrestin mobilization as defined herein; and

  Group 3 ISVDs or polypeptides can be characterized by binding / recognizing “part 1” epitopes (parts) as well as “group 2” epitopes (parts). Group 3 ISVDs or polypeptides are represented by 07B11 and are clearly between Group 1 and Group 2.

  Those skilled in the art are familiar with methods common in the art for determining epitopes (such as those provided in Example 11: “epitope mapping” of the present invention).

  Thus, the present invention relates to the polypeptide ISVD, and (conventional) antibodies, or portions thereof (eg Fc, Fab, Mini body).

  The anti-CXCR7 / CXCR7 bispecific constructs described above can be appropriately extended in half-life (eg pegylation, fusion to serum albumin, or peptides capable of binding to serum proteins such as serum albumin) Or, as further described herein, by fusion to a binding unit), thus, eg, a serum albumin binding peptide or bond, optionally linked via one or more suitable spacers or linkers Domains (such as those described herein) may further be included.

Again, such additional binding domains, binding units, amino acid sequences, or other (functional) groups or moieties may contain one or more other immunoglobulin single variable domains, such as one or more (single) ) Domain antibodies, dAbs or Nanobodies (eg, V _HH , humanized V _HH or camelized VH, eg camelized human VH) to provide a “bispecific” protein or polypeptide of the invention (ie, at least One, eg, one or two immunoglobulin single variable domains directed against CXCR7 and at least one, eg, one or two, immunoglobulin singles directed against another target Polypeptides of the invention comprising variable domains).

  For example, according to certain but non-limiting aspects, the constructs, proteins, or polypeptides of the present invention may have an increased half-life, for example, by functionalization and / or during construction, half of the construct. It may have been provided by including moieties or binding units that increase the phase. Examples of such functionalizations, moieties, or binding units will be apparent to those skilled in the art, but can be, for example, PEGylated, fused to serum albumin, or bound to a serum protein (such as serum albumin). It may include a fusion to a peptide or binding unit.

  The latter construct (ie, peptide or bond capable of binding to at least one, eg one or two, amino acid sequences of the invention and at least one, eg one or two, serum proteins such as serum albumin etc. In a fusion construct comprising units), the serum albumin binding peptide or binding domain can be any suitable serum albumin binding peptide or binding domain that is capable of increasing the half-life of the construct (serum albumin binding peptide or As compared to the same construct without binding domain), in particular as described by the applicant in WO 2008/068280 (and in particular in WO 2009/127691 and WO 2011/095545, both by the applicant) Serum albumin binding peptide, or a single variable domain of serum albumin binding immunoglobulin (eg, serum albumin binding Nanobody; eg, Alb-1 or a humanized version of Alb-1, such as Alb-8, etc. Reference is made, eg, to WO 06/122787).

  With respect to half-life, the serum albumin binding peptides according to WO 2008/068280, WO 2009/127691, and / or WO 2011/095545 are suitably used by using the various half-life extending techniques described herein. The half-life of the construct or polypeptide of the present invention is (preferably) suitably “matched” for the intended (therapeutic and / or diagnostic) application, and / or desired It should be noted in the present invention that the best balance can be obtained between therapeutic and / or pharmacological effects and possible unwanted side effects.

  Thus, for example, without limitation, a preferred aspect of the present invention is that a single variable domain of one immunoglobulin directed against human CXCR7 (or alternatively, two directed against human CXCR7 A single variable domain of an immunoglobulin (which may be the same or different), a “bivalent” polypeptide of the invention if they are the same or different, or a “biparental” polypeptide of the invention if they are different And a "bispecific" consisting essentially of a single variable domain of immunoglobulins (linked by a peptide linker) (as defined herein) directed against human serum albumin A polypeptide is provided, each providing a bispecific polypeptide of the invention (all as described herein). Such a protein or polypeptide can also be in substantially isolated form (as defined herein).

  In another specific but non-limiting aspect, the amino acid sequence of the invention (such as a Nanobody) or the polypeptide of the invention (such as a bivalent, biparental, or bispecific polypeptide of the invention). Can be appropriately linked to a toxin or to (cyto) toxic residues, moieties, or payload (again chemically or via one or more suitable linkers or spacers). For example, suitable (ie, an antibody-drug conjugate or “ADC” based on an amino acid sequence or polypeptide of the invention) can be linked to an amino acid sequence or polypeptide of the invention to provide a cytotoxic compound (ie, an amino acid sequence or polypeptide of the invention). Examples of (cell) damaging moieties, compounds, payloads, or residues will be apparent to those skilled in the art. References are made, for example, in a review by Ducry and Stump, Bioconjugate Chem., 2010, 21 (1), pp. 5-13. Such cytotoxic amino acid sequences or polypeptides of the invention, in particular, kill cells expressing the target to which the amino acid sequence or polypeptide of the invention is directed (eg in the treatment of cancer) or such It may be useful / suitable for those applications intended to reduce or slow cell growth and / or proliferation. Usually, but without limitation, the (cyto) toxic polypeptides of the present invention have either no extended half-life or only a limited and / or tightly controlled half-life extension. Yes.

  In another aspect, at least one amino acid sequence of the invention (ie, an immunoglobulin single variable domain for CXCR7) is suitably linked to at least one immunoglobulin single variable domain directed against CXCR4. There are also provided bispecific polypeptides of the invention directed against both CXCR7 and CXCR4.

  For example, in this aspect, at least one (eg, 1 or 2) amino acid sequence of the invention is suitable for an immunoglobulin single variable domain for at least one (eg, 1 or 2) CXCR4 You may connect to.

Some preferred but non-limiting examples of immunoglobulin single variable domains for CXCR4 that can be used in such constructs are the following (or may be chosen more appropriately below): :
A single variable domain of an immunoglobulin for CXCR4 (particularly one of the Nanobodies) from international application WO 09/138519 by Ablynx NV (for example, without limitation, in Table B-1.1 of WO 09/138519) 238D2 / SEQ ID NO: 238 and 238D4 / SEQ ID NO: 239); and / or-sequence optimization of the amino acid sequences 238D2 and 238D4 described in the unpublished US application filed June 25, 2010 by Ablynx NV Improved variants; and / or Ablynx NV., Filed Oct. 4, 2010. A single variable domain of an immunoglobulin capable of binding to the same epitope as 238D2 and / or 238D4 as described in PCT application PCT / EP2010 / 064766 by; and / or-filed January 7, 2011 10E9 type sequence, 281E10 type sequence, 10E12 type sequence, 10A10 type sequence, 10G10 type sequence, 14A2 type sequence, 15A1 type sequence described in PCT application PCT / EP2011 / 050156 by Ablynx NV 15H3 type sequence, and / or 283B6 type sequence; and / or-Ablynx NV. 10E9 type sequence, 281E10 type sequence, 10E12 type sequence, 10A10 type sequence, 10G10 type sequence, 14A2 type sequence, 15A1 type sequence, 15H3 type sequence described in PCT application PCT / EP2011 / 050156, pages 15-47, And / or 283B6 type sequences.

  The anti-CXCR7 / CXCR4 bispecific constructs described above (as well as other bispecific constructs comprising at least one amino acid sequence of the invention) can be suitably extended in half-life (eg, PEGylated, As described further herein) by fusion to serum albumin or by fusion to a peptide or binding unit capable of binding to a serum protein, such as serum albumin), thus, for example, optionally one or It may further comprise a serum albumin binding peptide or binding domain (such as those described herein) linked via a plurality of suitable spacers or linkers.

  Thus, one particular but non-limiting aspect of the invention is that one or two (preferably one) immunoglobulin single variable domains for CXCR7 (as defined herein) Preferably one or two Nanobodies), one or two (preferably one) immunoglobulin single variable domains for CXCR4 (preferably one or two Nanobodies as defined herein), and A polypeptide comprising a peptide or immunoglobulin single variable domain (optionally linked via one or more suitable spacers or linkers) to (human) serum albumin.

  The above anti-CXCR7 / CXCR4 bispecific constructs (as well as other bispecific constructs comprising at least one amino acid sequence of the invention) can also be added to toxins or to (cyto) toxic residues, moieties or payloads ( Appropriate linkages (as described further herein) (again chemically or via one or more suitable linkers or spacers). Again, such (cyto) toxic bispecific polypeptides of the present invention have no half-life, or only a limited and / or strictly controlled half-life extension. sell.

  The present invention, in its broadest sense, also includes derivatives of the amino acid sequences of the invention (eg, Nanobodies) and of the polypeptides of the invention. Such derivatives are generally obtained by modification of one or more of the amino acid sequences of the invention (eg, Nanobodies) and the polypeptides of the invention and / or the amino acid residues that form the Nanobodies of the invention, In particular, it can be obtained by chemical and / or biological (eg enzymatic) modification.

  Examples of such modifications, as well as amino acid sequences (eg, Nanobodies) and polypeptides of the invention that can be modified in such a manner (ie, either on the protein backbone, but preferably on the side chain) Examples of amino acid residues within, methods and techniques that can be used to introduce such modifications, and potential uses and advantages of such modifications will be apparent to those skilled in the art.

  For example, such modifications can include one or more functional groups, residues, or moieties in or on the amino acid sequences of the invention (eg, Nanobodies) and polypeptides of the invention, and in particular Introduction of one or more functional groups, residues, or moieties that confer one or more desired properties or functionality into the Nanobody of the invention (eg, by covalent linkage or another suitable In the form). Examples of such functional groups will be apparent to those skilled in the art.

  For example, such modifications increase any half-life, solubility and / or absorption of the Nanobodies of the invention, reduce the immunogenicity and / or toxicity of the Nanobodies of the invention, and any desired One or more functional groups (e.g., which eliminate or attenuate non-adverse side effects and / or confer other advantageous properties to the Nanobodies and / or polypeptides of the invention and / or reduce their undesired properties) By covalent linkage or in another suitable manner); or may include the introduction of any combination of the preceding. Examples of such functional groups and techniques for introducing them will be apparent to those skilled in the art, but generally all functionalities referred to in the general background art cited above in this specification. Groups and techniques, and may include functional groups and techniques known per se, for the modification of pharmaceutical proteins and in particular for the modification of antibodies or antibody fragments (including ScFv and single domain antibodies) However, for them, references are made, for example, to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may be linked directly (eg, covalently) to, for example, a Nanobody of the present invention, or optionally via a suitable linker or spacer, and again by those skilled in the art. As will be apparent.

  One of the most widely used techniques for increasing the half-life of pharmaceutical proteins and / or reducing immunogenicity is the use of suitable pharmacologically acceptable polymers such as poly (ethylene glycol) (PEG ) Or a derivative thereof (such as methoxypoly (ethylene glycol) or mPEG). In general, any suitable form of pegylation can be used (eg, in the art for antibodies and antibody fragments, including but not limited to (single) domain antibodies and ScFv). PEGylation used); see, for example, Chapman, Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Made in Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA.

  Preferably, site-specific pegylation is used, in particular via cysteine residues (see eg Yang et al., Protein Engineering, 16, 10, 761-770 (2003)). For example, for this purpose, PEG may be attached to a cysteine residue that occurs naturally in the Nanobody of the invention, and the Nanobody of the invention may be modified to leave one or more cysteine residues for attachment of PEG. A group may be introduced appropriately, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N and / or C terminus of the Nanobody of the invention, All use protein manipulation techniques known per se to those skilled in the art.

  Preferably, for Nanobodies and proteins of the invention, PEG is used and is greater than 5000, such as greater than 10,000 and less than 200,000, such as less than 100,000; for example, in the range of 20,000-80,000 With medium molecular weight.

  Another, usually less preferred modification, includes N-linked or O-linked glycosylation, usually as part of a translational and / or post-translational modification, and is used to express a Nanobody or polypeptide of the invention. Depends on the host cell.

  Yet another modification may involve the introduction of one or more detectable labels or other signal generating groups or moieties, depending on the intended use of the labeled Nanobody. Suitable labels and techniques for attaching, using, and detecting them will be apparent to those skilled in the art, but include, but are not limited to, fluorescent labels, phosphorescent labels, chemiluminescent labels, bioluminescent labels, Includes radioisotopes, metals, metal chelates, metal cations, chromophores, and enzymes (such as those mentioned on page 109 of WO 08/020079). Other suitable labels will include those moieties that can be detected using, for example, NMR or ESR spectroscopy, as will be apparent to those skilled in the art.

  Such labeled Nanobodies and polypeptides of the invention can be used in vitro, in vivo, or in situ assays (eg, immunoassays known per se, such as ELISA, RIA, EIA, and other "depending on the choice of the particular label"). Etc.) as well as in vivo diagnostic and imaging purposes.

  As will be apparent to those skilled in the art, another modification may involve the introduction of a chelating group, but, for example, chelate one of the metals or metal cations referred to above. Suitable chelating groups include, without limitation, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

  Yet another modification may involve the introduction of a functional group that is a part of a specific binding pair, such as a biotin (strept) avidin binding pair. Using such a functional group, the Nanobody of the present invention can be linked to another protein, polypeptide, or chemical compound that is bound to the other half of the binding pair, ie, through the formation of the binding pair. Also good. For example, a Nanobody of the invention may be conjugated to biotin and linked to another protein, polypeptide, compound, or carrier (conjugated to avidin or streptavidin). For example, such conjugated Nanobodies may be used as reporters, for example, in diagnostic systems, where a detectable signal producing agent is conjugated to avidin or streptavidin. Such binding pairs may also be used, for example, to bind the Nanobodies of the present invention to a carrier, including a carrier suitable for pharmaceutical purposes. One non-limiting example is the liposomal formulation described by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs may also be used to link therapeutically active agents to the Nanobodies of the present invention.

  Other potential chemical and enzymatic modifications will be apparent to those skilled in the art. Such modifications can also be introduced for research purposes (eg, to test function-activity relationships). References are made, for example, to Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

  The immunoglobulin single variable domains and polypeptides of the invention are as such, preferably not linked to any other amino acid sequence or chain via a disulfide bridge (but one or more). Consisting essentially of a single amino acid chain). For example, it is known that the agents of the present invention (as described herein) can sometimes contain a disulfide bridge between CDR3 and CDR1 or FR2. However, one or more immunoglobulin single variable domains of the invention are linked to each other and / or to other immunoglobulin single variable domains (eg, via disulfide bridges), It should be noted that peptide constructs (eg, Fab ′ fragments, F (ab ′) 2 fragments, ScFv constructs, “diabodies”, and other multispecific constructs) that may be useful may be provided. References are made, for example, to the review article by Holliger and Hudson, Nat Biotechnol. 2005 Sep; 23 (9): 1126-36.

  In general, the amino acid sequences of the invention (or compounds, constructs, or polypeptides comprising the same) are for therapeutic and / or diagnostic purposes for administration to a subject (eg, as described herein). If intended (for technical purposes), it is preferably an amino acid sequence that does not naturally occur in the subject; or, if it occurs naturally in the subject, a substantially isolated form (herein As defined).

  Also, for pharmaceutical use, the immunoglobulin single variable domains of the present invention (as well as compounds, constructs and polypeptides comprising the same) are preferably against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). For veterinary purposes, the immunoglobulin single variable domains and polypeptides of the invention are preferably directed against CXCR7 from the species to be treated, Alternatively, it will be apparent to one skilled in the art that it is at least cross-reactive with CXCR7 from the species to be treated.

  Furthermore, the amino acid sequences of the present invention optionally comprise 1 for binding to other antigens, proteins or targets, in addition to at least one binding site for binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). Contains one or more additional binding sites.

  The efficacy of the immunoglobulin single variable domains and polypeptides of the invention, and compositions comprising the same, depending on the particular disease or disorder involved, any suitable in vitro assay, cell-based assay, In vivo assays and / or animal models known per se, or any combination thereof, can be used for testing. Suitable assays and animal models will be apparent to those skilled in the art, for example, ligand displacement assays (Burns et al, J. Exp. Med. 2006 4; 203 (9): 2201-13), beta arrestin mobilization assays (Zabel et al., J. Immunol. 2009 1; 183 (5): 3204-11), dimerization assay (Luker et al, Faseb J. 2009 23 (3): 823-34), signal transduction assay (Wang et al, J Immunol. 2009 Sep 1; 183 (5): 3204-11), proliferation assay (Wang et al, J Immunol. 2009 Sep 1; 183 (5): 3204-11; Odemis et al., J Cell Sign. 2010 April 1; 123 (Pt 7): 1081-8), survival assay (Burns et al, J. Exp. Med. 2006 4; 203 (9): 2201-13), cell adhesion assay (Burns et al, J. Exp. Med. 2006 4; 203 (9): 2201-13) and transendothelial migration assay (Mazzinghi et al, J. Exp. Med. 2008 Feb 18; 205 (2): 479-90) , Endothelial cell germination assay (Wang et al, J Immunol. 2009 Sep 1; 183 (5): 3204-11), muscle differentiation (Melchionna et al., Muscle Nerve, 2010 Feb 11) and In vivo xenograft model (Burns et al, J. Exp. Med. 2006 4; 203 (9): 2201-13), collagen-induced arthritis model (Hegen et al, Ann Rheum Dis. 2008 Nov; 67 (11) : 1505-15), and experimental autoimmune encephalomyelitis model (Wekerle, Ann Rheum Dis. 2008 Dec; 67 Suppl 3: iii56-60) and the following experimental part and prior art cited herein Assays and animal models.

  Also according to the present invention, an immunoglobulin single variable domain and polypeptide directed against CXCR7 from a first species of warm-blooded animal is CXCR7 from one or more other species of warm-blooded animal. May or may not show cross-reactivity. For example, immunoglobulin single variable domains and polypeptides directed against human CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) are primates (eg, without limitation, monkeys from the genus Macaque (eg, , CXCR7 from one or more species of cynomolgus monkeys (such as Macaca fascicularis) and / or rhesus monkeys (such as Macaca mulatta) and baboons (such as Papioursinus) and / or animal models for disease (eg, mice, In rats, rabbits, pigs or dogs), particularly in animal models for diseases and disorders associated with human CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), such as the species and animal models referred to herein. Often used movement It would not show may or exhibit CXCR7 cross-reactive with one or more species of. In this regard, it will be apparent to those skilled in the art that such cross-reactivity, if present, can have advantages from a drug development perspective. This is because it allows immunoglobulin single variable domains and polypeptides against human CXCR7 and especially human CXCR7 (SEQ ID NO: 1) to be tested in such disease models (eg, Example 12). checking).

  More generally, immunoglobulin single variable domains and polypeptides of the invention that are cross-reactive with CXCR7 from multiple species of mammals are generally advantageous for use in veterinary applications. It is possible. This is because it allows the same amino acid sequence or polypeptide to be used across multiple species. Thus, an immunoglobulin single variable domain and polypeptide directed against CXCR7 from one species of animal, such as an immunoglobulin single variable domain and polypeptide for human CXCR7 (SEQ ID NO: 1), etc. Can also be used in the treatment of other species of animals, so long as the use of a single variable domain and / or polypeptide of an immunoglobulin provides the desired effect in the species to be treated. Is included in the range.

  The present invention, in its broadest sense, is not particularly limited, or specific antigenic determination of CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) to which the immunoglobulin single variable domains and polypeptides of the invention are directed. Defined by group, epitope, portion, domain, subunit, or conformation (if applicable). For example, immunoglobulin single variable domains and polypeptides may include CXCL11 / CXCL12 interaction sites and / or CXCR7 / CXCR7 homodimerization sites and / or CXCR4 / CXCR7 heterodimerization sites (or CXCR7 and others). Chemokine receptors such as, for example, heterodimerization to CXCR3, etc., may or may not be directed, as further defined herein.

  As further described herein, a polypeptide of the invention may comprise a single variable domain of two or more immunoglobulins of the invention directed against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). . In general, such polypeptides can bind to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) with increased avidity compared to the single amino acid sequence of the present invention. Such polypeptides are directed, for example, to the same antigenic determinant, epitope, portion, domain, subunit, or conformation (if applicable) of CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) Which may comprise a single variable domain of two immunoglobulins of the present invention (which may or may not be the interaction site); alternatively, the first of CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) At least one “first” amino acid sequence of the invention directed to one and the same antigenic determinant, epitope, portion, domain, subunit, or conformation (where applicable) May or may not be the site of interaction) (eg group 1 As well as a second antigenic determinant, epitope, portion, domain, subunit, or conformation (if applicable) of the present invention that is different from the first It comprises at least one “second” amino acid sequence, which may or may not be an interaction site again (eg, a group 2 epitope, etc.). Preferably, in such “biparental” polypeptides of the invention, at least one amino acid sequence of the invention is directed against the interaction site (as defined herein) The invention is not limited thereto in its broadest sense. For example, the polypeptides of the invention may be formatted, eg, in a biparental method, combining monovalent building blocks directed against different epitopes as characterized in the experimental part (Example 9 ~ 17). The binding constant (eg, association and dissociation constant) of an individual immunoglobulin of a “bivalent” polypeptide is the binding constant of an individual immunoglobulin single variable domain of a “biparental” polypeptide. Although generally better, the present invention is completely unexpected that the “biparental” polypeptide of the present invention is a “bivalent” polypeptide in biological assays (eg, β-arrestin assays). To prove more effective.

  Alternatively, if the target is part of a binding pair (eg, a receptor-ligand binding pair), an immunoglobulin single variable domain and polypeptide may be associated with a cognate binding partner, eg, CXCL11 (also I- And / or CXCL12 (also referred to as SDF-1) to compete for binding to CXCR7 and / or they bind the binding partner to the target ( Neutralize completely or partially).

  Also, the immunoglobulin single variable domains and polypeptides of the present invention generally comprise all naturally occurring or synthetic analogs, variants, variants, alleles of CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). Or at least a single variable domain and polypeptide of an immunoglobulin of the invention is substantially the same as an antigenic determinant or epitope that binds to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). It is predicted to bind to CXCR7 and in particular their analogs, variants, variants, alleles, portions, and fragments of human CXCR7 (SEQ ID NO: 1), including one or more antigenic determinants or epitopes . Again, in such cases, the single variable domains and polypeptides of the immunoglobulins of the invention may be converted to such analogs, variants, variants, alleles, portions and fragments of the immunoglobulins of the invention. One variable domain may bind with the same affinity and specificity as binding to (wild-type) CXCR7, or with a different (ie higher or lower) affinity and / or specificity.

  CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) are in monomeric form and in one or more multimeric forms, eg in homodimeric form, and also in CXCR4, eg human CXCR4 (RM Maksym et al., supra; KE Luker et al., supra) when present in a heterodimeric form, the immunoglobulin single variable domains and polypeptides of the invention are i) a monomeric form of CXCR7 and Binds specifically to human CXCR7 (SEQ ID NO: 1) only, ii) binds only to multimeric / dimeric (homo and / or heterodimer) forms of CXCR7 and particularly human CXCR7 (SEQ ID NO: 1), or iii It is within the scope of the present invention to bind to both monomeric and multimeric forms. In a preferred aspect of the invention, the polypeptides of the invention prevent the formation of homodimeric human CXCR7 complexes and / or heterodimeric human CXCR4 / CXCR7 complexes. In another preferred aspect of the invention, the polypeptides of the invention do not induce the formation of homodimeric human CXCR7 complex and / or heterodimeric human CXCR4 / CXCR7 complex (even at higher concentrations, for example 10 nM or less, 50 nM or less, 100 nM or less, or 500 nM or less). Again, in such cases, the polypeptide of the invention is in monomeric form, with the same affinity and specificity that the single variable domain of the immunoglobulin of the invention binds to the multimeric form, or , May bind with a different (ie higher or lower) affinity and / or specificity.

  Also, CXCR7 and particularly human CXCR7 (SEQ ID NO: 1) may associate with other proteins or polypeptides to form protein complexes (eg, using CXCL12 / SDF-1 or CXCL11 / I-TAC). Where possible, immunoglobulin single variable domains and polypeptides of the invention bind to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) in its unassociated state (eg, prevent ligand binding), CXCR7 and in particular It is within the scope of the present invention to bind to human CXCR7 (SEQ ID NO: 1) in its associated state, or to both (preferably in a non-associated state). In all these cases, the immunoglobulin single variable domains and polypeptides of the invention are in such an associated protein complex and the immunoglobulin single variable domains and polypeptides of the invention are in their unassociated state. May bind with affinity and / or specificity that may be the same or different (ie, higher or lower) than the affinity and specificity that binds to CXCR7 and particularly human CXCR7 (SEQ ID NO: 1).

  Also, as will be apparent to those skilled in the art, proteins or polypeptides comprising two or more immunoglobulin single variable domains directed against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) are CXCR7 and in particular human. It can bind to CXCR7 (SEQ ID NO: 1) with a higher binding force than the corresponding monomeric amino acid sequence. For example, without limitation, a protein or polypeptide comprising two or more immunoglobulin single variable domains directed against different epitopes of CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) may be of different monomers A protein or poly that can bind (usually) with a higher binding force than each, and contains two or more immunoglobulin single variable domains directed against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) Peptides are also associated with higher avidity, with CXCR7 multimers (eg, homodimers, heterodimers with CXCR4) and especially human CXCR7 (SEQ ID NO: 1) multimers (eg, homodimers). Can be (usually) bound to a monomer, a heterodimer with human CXCR4.

  In general, the single variable domains and polypeptides of the immunoglobulins of the present invention are those of CXCR7 and the particular human CXCR7 (SEQ ID NO: 1) most relevant from at least a biological and / or therapeutic point of view. (Including monomers, multimers and associated different conformational forms) as will be apparent to those skilled in the art.

  Also, using single immunoglobulin variable domain and polypeptide portions, fragments, analogs, variants, variants, alleles and / or derivatives of the invention and / or such portions, fragments It is contemplated herein to use a protein or polypeptide comprising, or consisting essentially of, one, more than one, analog, variant, variant, allele, and / or derivative As long as it is suitable for use, it is within the scope of the present invention. Such portions, fragments, analogs, variants, variants, alleles, and / or derivatives are usually at least a portion of a functional antigen binding site for binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). More preferably, specific binding to CXCR7 and special human CXCR7 (SEQ ID NO: 1) is possible, even more preferred binding to CXCR7 and special human CXCR7 (SEQ ID NO: 1) As described further herein, for example, in the experimental part defined herein, EC50 values, mean Ki, IC50 values for binding, migration, displacement, and / or growth blockade, and / or Or with other measures of potency, such parts, fragments, analogs, variants, variants, Allelic, and / or derivatives, more potent, more stable, can be a more soluble, and may have the same epitope. Some non-limiting examples of such portions, fragments, analogs, variants, variants, alleles, derivatives, proteins, and / or polypeptides will become clear from the further description herein. . Additional fragments or polypeptides of the present invention are also provided by appropriate combination (ie, by ligation or gene fusion) of one or more (smaller) portions or fragments as described herein. sell.

For a general description of immunoglobulin single variable domains, references are made to the further description below as well as to the prior art cited herein. In this regard, however, this description and the prior art are mainly based on the so-called “V _H 3 class” immunoglobulin single variable domain (ie, a high degree of sequence homology with the V _H 3 class human germline sequence). It should be noted that it describes a single variable domain of an immunoglobulin with, such as DP-47, DP-51, or DP-29, which forms a preferred aspect of the present invention. However, the present invention, in its broader sense, is generally directed to any type of immunoglobulin single variable domain directed against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), for example A single immunoglobulin domain belonging to the so-called “V _H 4 class” (ie, a single variable domain of an immunoglobulin with a high degree of sequence homology with a human germline sequence of the V _H 4 class, such as DP-78). It should be noted that one variable domain is targeted (eg as described in WO 07/118670).

In general, immunoglobulin single variable domains (especially V _HH sequences and sequence-optimized immunoglobulin single variable domains) are in particular framework sequences (again, as further described herein). Can be characterized by the presence of one or more “hole mark residues” (as described herein) in one or more of the above.

Thus, in general, an immunoglobulin single variable domain can be defined as an amino acid sequence with the following (generic) structure:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and here, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively.

In a preferred aspect, the present invention provides a polypeptide comprising at least a single variable domain of an immunoglobulin that is an amino acid sequence with the following (generic) structure:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and here, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and where:
i) At least one of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering is the hole mark residue referred to in Table A-1 below. Selected from the group. And / or where:
ii) the amino acid sequence is at least 80% and more preferably 90% with at least one of the immunoglobulin single variable domains as shown in WO 2009/138519 (see SEQ ID NO: 1-125 in WO 2009/138519); %, Even more preferably 95% amino acid identity, where for the purpose of determining the degree of amino acid identity, the amino acid residues forming the CDR sequence (denoted with X in the sequence) ); And / or where:
iii) CDR sequences are generally as further defined herein (eg, CDR1, CDR2, and CDR3 in combination as provided in Table B-2. CDR definitions are defined as Kabat numbering) Note that it is calculated according to the system); and / or where:
iv) The FR sequence is generally as further defined herein, eg, FR1, FR2, FR3, and FR4 in combination, as provided in Table B-2, and / or FR1, FR2, FR3, and FR4, as provided in Table B-2, are at least 80%, more preferably 90%, and even more preferably at least one of FR1, FR2, FR3, and FR4, respectively, of FR. Has 95% amino acid identity (where the FR definition is calculated according to the Kabat numbering system).

Again, such immunoglobulin single variable domains can be derived in any suitable manner and from any suitable source, eg, naturally occurring V _HH sequences (ie, suitable for camels) Species, eg from llamas) or synthetic or semi-synthetic V _H or VL (eg from humans). Such immunoglobulin single variable domains include “humanized” or other “sequence-optimized” VHH, “camelized” immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences, ie Camelized VH), as well as techniques such as affinity maturation (eg, starting with synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments from different immunoglobulin sequences , PCR assembly using overlapping primers, and similar techniques for manipulating immunoglobulin sequences well known to those skilled in the art; or any suitable combination of any of the preceding (as further described herein) ), Etc., which may be altered by human VH, human VL, camel VHH.

  In a further preferred aspect, the present invention relates to one immunoglobulin with an amino acid sequence selected from the group consisting of amino acid sequences with SEQ ID NOs: 39-43 and 91 and 99-102 (see Table B-3). Provided are polypeptides comprising an immunoglobulin single variable domain with an amino acid sequence selected from the group consisting of a single variable domain and a moiety that provides an increased half-life (see below).

In a further preferred aspect, the present invention provides an amino acid sequence selected from the group consisting of an amino acid sequence substantially consisting of four framework regions (respectively FR1 to FR4) and three complementarity determining regions (respectively CDR1 to CDR3). A polypeptide comprising at least one immunoglobulin single variable domain is provided, wherein the CDR sequences of said amino acid sequences are the immunoglobulin single variable domains of SEQ ID NOs: 39-43 and 91 and 99-102. At least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as greater than 95% amino acid identity, or even more substantial 100% amino acid identity (Table B-2 and See -3). This degree of amino acid identity is, for example, between the amino acid sequence and one or more of the sequences of SEQ ID NOs: 39-43 and 91 and 99-102 (see Tables B-2 and B-3). It can be determined by determining the degree of amino acid identity (in the manner described herein) where the amino acid residues that form the framework regions are ignored. Such polypeptide and / or immunoglobulin single variable domains of the invention may further provide:
1. Directed against CXCR7 and especially human CXCR7 (SEQ ID NO: 1) (as defined herein), and SEQ ID NOs: 39-43 and 91 and 99-102 (see Table B-3) A polypeptide comprising at least one immunoglobulin single variable domain having at least 80%, preferably at least 85%, such as 90% or 95% sequence identity with an immunoglobulin single variable domain;
2. Directed against CXCR7 and specifically human CXCR7 (SEQ ID NO: 1) (as defined herein), and SEQ ID NOs: 39-43 and 91 and 99 to CXCR7 and particularly human CXCR7 (SEQ ID NO: 1) Cross-block (as defined herein) and / or SEQ ID NOs: 39-43 and 91 at least one binding of an immunoglobulin single variable domain of ˜102 (see Table B-3) And at least one of 99 to 102 (see Table B-3) of immunoglobulin single variable domains that compete for binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). 2. a polypeptide comprising a single variable domain; The immunoglobulin single variable domain may be as further described herein; and a polypeptide of the invention comprising one or more of such immunoglobulin single variable domains ( As described further herein, for example, bispecific (eg, can also be binding to serum albumin) and / or biparental polypeptides), and such immunoglobulins A nucleic acid sequence encoding a variable domain and polypeptide. Such immunoglobulin single variable domains and polypeptides do not include any naturally occurring ligands.

  The polypeptides of the invention comprise or consist essentially of at least one immunoglobulin single variable domain of the invention. Some preferred but non-limiting examples of immunoglobulin single variable domains of the invention are given in SEQ ID NOs: 39-43 and 91 and 99-102 (see Table B-3).

1.2. In other building blocks another aspect of increasing the serum albumin binding building blocks or half-life, the present invention is human CXCR7 (or a suitable fragment) one directed or more (preferably one) immunization A compound or construct, and in particular a protein or poly, comprising or consisting essentially of a single variable domain of a globulin and optionally further comprising one or more other groups, residues, moieties or binding units Peptides (also referred to herein as “compounds of the invention” or “polypeptides of the invention”, respectively). As will be apparent to those skilled in the art from further disclosure herein, such additional groups, residues, moieties, binding units, or single variable domains of immunoglobulins may provide additional functionality for the amino acids of the invention. The sequence (and / or the compound or construct in which it is present) may or may not be provided, and the properties of the amino acid sequence of the present invention may or may not be modified.

  As can be seen from the further description above and herein, this is because the single variable domains of the immunoglobulins of the present invention serve as “building blocks” to form the polypeptides of the present invention, ie, they Can be combined with other groups, residues, moieties, or binding units, as appropriate, to form a compound or construct as described herein (e.g., without limitation, of the invention described herein). Can be used to form one or more desired properties or biologicals within a molecule. Combine functions.

  A compound or polypeptide of the present invention generally comprises a single variable domain of one or more immunoglobulins of the present invention, optionally in one or more additional groups, residues, moieties or binding units. It can be prepared by a method comprising at least one step of appropriately linking through one or more suitable linkers to provide a compound or polypeptide of the invention. The polypeptides of the invention also generally include at least the steps of providing a nucleic acid encoding the polypeptide of the invention, expressing the nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. . Such methods can be carried out in a manner known per se, which will be clear to the person skilled in the art, for example based on the methods and techniques described further herein.

  The process of designing / selecting and / or preparing a compound or polypeptide of the present invention begins with an amino acid sequence of the present invention and is referred to herein as “formatting” the amino acid sequence of the present invention; And the amino acids of the present invention that make up part of the compound or polypeptide of the present invention are said to be “formatted” or “during formatting” of said compound or polypeptide of the present invention. Examples of methods by which the amino acid sequences of the present invention can be formatted and examples of such formats will be apparent to those of skill in the art based on the disclosure herein; such formatted immunoglobulin singles Variable domains form a further aspect of the invention.

  For example, such additional groups, residues, moieties, or binding units can be a single variable domain of one or more additional immunoglobulins, but the compound or construct is a (fusion) protein or (fusion) poly Be a peptide. In a preferred but non-limiting aspect, the one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, residues, moieties or binding units are a domain antibody, a single variable domain of an immunoglobulin suitable for use as a domain antibody, a single Consisting of a domain antibody, an immunoglobulin single variable domain suitable for use as a single domain antibody, a “dAb”, an immunoglobulin single variable domain suitable for use as a dAb, or a Nanobody Selected from the group. Alternatively, such groups, residues, moieties or binding units may be, for example, chemical groups, residues, moieties, which are themselves biologically and / or pharmacologically. May or may not be active. For example, without limitation, such groups may be linked to a single variable domain of one or more immunoglobulins of the present invention, providing “derivatives” of the amino acid sequences or polypeptides of the present invention. (As described further herein).

  Also within the scope of the invention is a compound or construct, which comprises or consists essentially of one or more derivatives as described herein, and optionally It further includes one or more other groups, residues, moieties, or binding units linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are immunoglobulin single variable domains. In the compounds or constructs described above, one or more immunoglobulin single variable domains and one or more groups, residues, moieties or binding units of the invention may be combined with each other and / or one Alternatively, they may be linked directly via multiple suitable linkers or spacers. For example, if one or more groups, residues, moieties, or binding units are an immunoglobulin single variable domain, the linker can also be an immunoglobulin single variable domain, but the resulting The compound or construct should be a fusion (protein) or a fusion (polypeptide).

  In one specific but non-limiting aspect of the present invention, it may be further described herein, the polypeptides of the present invention are compared to the single variable domain of the immunoglobulin from which they are derived. And has an increased half-life in serum (as described further herein). For example, a single variable domain of an immunoglobulin of the invention may be linked (chemically or otherwise) to one or more groups or moieties (such as PEG) that increase the half-life, and have an increased half-life. A derivative of the amino acid of the present invention is provided.

  In one particular aspect of the invention, a compound of the invention or a polypeptide of the invention may have an increased half-life compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such compounds and polypeptides will be apparent to those skilled in the art based on the further disclosure herein, but may be chemically modified, for example. A single variable domain or polypeptide of an immunoglobulin of the invention having an increased half-life (eg, using pegylation); comprising at least one additional binding site for binding to serum proteins A single variable domain of an immunoglobulin of the invention; at least one amino acid sequence of the invention linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the amino acid sequence of the invention; Including the polypeptide of the present invention. Examples of polypeptides of the invention comprising such half-life extending moieties or immunoglobulin single variable domains will be apparent to those of skill in the art based on the further disclosure herein; Without a single variable domain of one or more immunoglobulins of the present invention is one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or serum proteins (such as A single variable domain of an immunoglobulin suitable for use as a domain antibody, domain antibody, single domain antibody, an immunoglobulin single variable domain suitable for use as a single domain antibody, " dAb ", a single variable domain of an immunoglobulin suitable for use as a dAb, or blood Nanobodies, serum immunoglobulins (such as IgG), or transferrins that can bind to proteins, such as serum albumin (such as human serum albumin); references are made to the further descriptions and references mentioned herein. A polypeptide suitably linked to one or more binding units capable of binding to the amino acid sequence of the present invention; and an amino acid sequence of the invention linked to an Fc portion (eg, human Fc, etc.) or a suitable portion or fragment thereof. Or one or more small proteins or peptides (eg, without limitation, one or more small proteins or peptides) capable of binding a single variable domain of one or more immunoglobulins of the invention to a serum protein. 91/01743, WO 01/45746, WO 02 / 76,489, including WO2008 / 068280, WO2009 / 127691 polypeptide is suitably coupled to the proteins and peptides, etc.) are described.

  In general, a compound or polypeptide of the invention with an increased half-life is preferably at least 1.5 times, preferably at least 2-fold, eg, at least twice the half-life of the corresponding amino acid sequence of the invention itself. It has a large half-life, such as at least 5 times, for example at least 10 times or more than 20 times. For example, a compound or polypeptide of the invention with an increased half-life is greater than 1 hour, preferably greater than 2 hours, more preferably 6 hours compared to the corresponding amino acid sequence of the present invention in humans. May have an increased half-life, such as greater than 12 hours, such as greater than 12 hours, or even greater than 24, 48, or 72 hours.

  In a preferred but non-limiting aspect of the invention, such a compound or polypeptide of the invention is more than 1 hour in human compared to the corresponding amino acid sequence of the invention itself, preferably Has an increased serum half-life of greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, or even greater than 24, 48, or 72 hours.

  In another preferred but non-limiting aspect, such a compound or polypeptide of the invention is at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferred in humans. Indicates a serum half-life of at least 72 hours or more. For example, a compound or polypeptide of the invention can be used for at least 5 days (eg, about 5-10 days), preferably at least 9 days (eg, about 9-14 days), more preferably at least about 10 days (eg, about 10-15). Day), or at least about 11 days (eg, about 11-16 days), more preferably at least about 12 days (eg, about 12-18 days or more), or more than 14 days (eg, about 14-19 days). Can be included.

  In certain preferred but non-limiting aspects of the invention, the present invention provides: i) a single variable domain of one CXCR7 binding immunoglobulin as described herein; and ii) Provided is a polypeptide of the invention comprising a single variable domain of one or more (preferably one) serum albumin binding immunoglobulin as described.

  In a further preferred aspect, the invention provides i) a single variable domain of one or more CXCR7 binding immunoglobulins as described herein; and ii) one of SEQ ID NO: 2 (Table B-1) or Provided are polypeptides of the invention comprising a single (preferably one) serum albumin binding immunoglobulin single variable domain.

  In a further preferred aspect, the invention provides a sequence comprising i) a single variable domain of one or more CXCR7 binding immunoglobulins as described herein; and ii) a CDR (defined according to Kabat numbering). Provided is a polypeptide of the invention comprising a single variable domain of one or more (preferably one) serum albumin binding immunoglobulin of number 2 (Table B-2, B-1).

  Thus, for example, further references (and thus incorporated by reference) are made in particular in the experimental part of WO 2008/068280 and further description, where further details regarding SEQ ID NO: 2 are made, For example, the half-life of an immunoglobulin single variable domain construct comprising the sequence in rhesus monkeys is disclosed.

  In general, a protein or polypeptide comprising or consisting essentially of a single variable domain of a single immunoglobulin is referred to herein as a “monovalent” protein or polypeptide or as a “monovalent construct”. Is done. A single variable domain of two or more immunoglobulins (eg, a single variable domain of at least two immunoglobulins of the invention or a single variable domain of at least one immunoglobulin of the invention and a single variable domain of at least one other immunoglobulin). Proteins and polypeptides comprising or consisting essentially of one variable domain, etc. may be referred to herein as “multivalent” proteins or polypeptides or as “multivalent constructs” Compared to a single variable domain of the corresponding monovalent immunoglobulin, it may provide certain advantages. Some non-limiting examples of such multivalent constructs will become clear from the further description herein.

  In accordance with another specific but non-limiting aspect, the polypeptides of the present invention comprise at least one immunoglobulin single variable domain and at least one other binding unit (ie, another epitope, antigen, target, (Which is preferably also a single variable domain of an immunoglobulin) or consists essentially of a protein, or polypeptide. Such proteins or polypeptides are also referred to herein as “multispecific” proteins or polypeptides or as “multispecific constructs”, which are the single immunoglobulins of the two immunoglobulins of the invention. It may consist of a variable domain, for example one immunoglobulin single variable domain directed against CXCR7 and one immunoglobulin single variable domain for serum albumin. Such multispecific constructs will be apparent to those of skill in the art based on the disclosure herein; some preferred, but not limited, of such multispecific immunoglobulin single variable domains Specific examples include constructs of SEQ ID NOs: 44-48, 80-81, 83-85, and 88-89 and 131-140 (see Table B-4), and clones 009, 013, 018-029, 031-038, 044, 046, 048-053, 055-058, 060, 061, 063, 065, 068, 069, 072, 081-086, and 093 (Tables B-12 to B-14).

  In accordance with yet another specific, but non-limiting aspect, the polypeptides of the present invention can comprise a single variable domain of at least one immunoglobulin of the present invention, optionally one or more additional immunoglobulin singles. A variable domain, and at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the single variable domain of the immunoglobulins of the invention and / or the resulting fusion protein. Or substantially consists of Again, such fusion proteins may provide certain advantages, eg, increased half-life, compared to the corresponding monovalent immunoglobulin single variable domain of the present invention.

  In the above construct, one or more immunoglobulin single variable domains and / or other immunoglobulin single variable domains can be directly linked to each other and / or one or more linker sequences to each other. Can be connected appropriately. Some suitable but non-limiting examples of such linkers will become clear from the further description herein.

  In one embodiment, the linker sequence linking immunoglobulin single variable domains is SEQ ID NO: 49-58 (see Table B-5), or a combination of both, or known in the art Street.

  In accordance with yet another specific, but non-limiting aspect, the polypeptides of the present invention are, for example, the immunoglobulin singles of SEQ ID NOs: 39-43 and 91 and 99-102 (see Table B-3). One or more of one variable domain and more than 80%, preferably more than 90%, more preferably more than 95%, eg 99% or more “sequence identity” (as defined herein) Wherein the polypeptide is preferably as further defined herein, i.e. one immunity directed against CXCR7. There is a preferred format for a single variable domain of globulin and a single variable domain of one immunoglobulin to one serum albumin.

  In accordance with yet another specific but non-limiting aspect, the polypeptide of the present invention is, for example, greater than 80%, preferably greater than 90% with one or more of the polypeptides of SEQ ID NOs: 44-48, More preferably, it may be selected from the group consisting of polypeptides having “sequence identity” (as defined herein) of greater than 95%, eg 99% or more (see Table B-4). Some exemplary, non-limiting examples of biparental and bispecific polypeptides of the invention include SEQ ID NOs: 78-89 and SEQ ID NOs: 131-140, or clones 009, 013, 018-029, 031. To 038, 044, 046, 048-053, 055-058, 060, 061, 063, 065, 068, 069, 072, 081-086, and 093 (Tables B-12 to B-14).

1.3. Compositions of the Invention Generally for pharmaceutical use, a polypeptide of the invention comprises at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent, or excipient. And / or an adjuvant, and optionally, may be formulated as a pharmaceutical preparation or composition comprising one or more additional pharmaceutically active polypeptides and / or compounds. Using non-limiting examples, such formulations are for oral administration, for parenteral administration (eg, by intravenous, intramuscular, or subcutaneous injection or intravenous infusion), for topical administration. It may be in a form suitable for administration by inhalation, by skin patch, by implant, by suppository, etc. Here, parenteral administration is preferred. Such suitable dosage forms (which can be solid, semi-solid, or liquid, depending on the mode of administration) and carriers for use in the methods and their preparations will be apparent to those skilled in the art. Are further described herein. Such pharmaceutical preparations or compositions will generally be referred to herein as “pharmaceutical compositions”. Pharmaceutical preparations or compositions for use in non-human organisms will generally be referred to herein as “veterinary compositions”.

  Thus, in a further aspect, the invention provides at least one amino acid of the invention, at least one polypeptide of the invention, or at least one polypeptide of the invention and at least one suitable carrier, diluent, or It relates to a pharmaceutical composition comprising an excipient (ie suitable for pharmaceutical use) and optionally one or more further active substances.

  In general, the polypeptides of the invention can be formulated and administered in any suitable manner known per se. Reference is made, for example, in the general background cited above (and in particular in WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867, and WO 08/020079) and in standard handbooks. For example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see, for example, pages 252-255).

  The polypeptides of the present invention can be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv and diabody) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be apparent to those skilled in the art, for example, parenteral administration (eg, intravenous, intraperitoneal, subcutaneous, intramuscular, intracavitary, intraarterial, or arachnoid) Preparations suitable for intraluminal administration) or for topical (ie transdermal or intradermal) administration.

  Preparations for parenteral administration can be, for example, sterile solutions, suspensions, dispersions, or emulsions suitable for infusion or injection. Suitable carriers or diluents for such preparations include, for example, without limitation, those mentioned on page 143 of WO 08/020079. In one embodiment, the preparation is an aqueous solution or suspension.

  The polypeptides of the invention can be administered using delivery gene therapy methods. See, for example, US Pat. No. 5,399,346. It is incorporated by reference for its gene therapy delivery method. Primary cells transfected with a gene encoding the amino acid sequence of the present invention, polypeptide, using a gene therapy method of delivery, are additionally transfected with a tissue-specific promoter to produce a specific organ, tissue Grafts, tumors, or cells can be targeted, and in addition can be transfected with signals and stabilizing sequences for localized expression in the cells.

  Thus, the polypeptides of the invention may be administered systemically, for example orally in combination with a pharmaceutically acceptable medium (such as an inert diluent or an assimilable edible carrier). . They can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For oral therapeutic administration, the polypeptides of the invention may be combined with one or more excipients, such as ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. It may be used in the form. Such compositions and preparations should contain at least 0.1% of the polypeptide of the invention. Their percentages in the compositions and preparations will, of course, vary, but can conveniently be between about 2 and about 60% of the weight of a given unit dosage form. The amount of the polypeptide of the invention in such therapeutically useful compositions is such that an effective dosage level is obtained.

  For topical administration at the site of tumor resection, the polypeptides of the invention may be used in biodegradable polymer drug delivery systems, sustained release poly (lactic acid) -co-glycolic acid formulations and the like (Hart et al. al., Cochrane Database Syst Rev. 2008 Jul 16; (3): CD007294).

  In a further preferred aspect of the invention, the polypeptide of the invention consists essentially of a single variable domain of a monovalent anti-human CXCR7 immunoglobulin and a single variable domain of a monovalent anti-human serum albumin immunoglobulin ( Polypeptides etc. (linked by a GS linker) etc. may have a beneficial distribution and kinetic profile in solid tumors compared to conventional antibodies (eg IgG etc.).

  Tablets, troches, pills, capsules, etc. may also contain binders, excipients, disintegrants, lubricants and sweetening or flavoring agents, such as those mentioned in pages 143 to 144 of WO 08/020079. . Where the unit dosage form is a capsule, it can contain, in addition to the above types of materials, a liquid carrier such as vegetable oil or polyethylene glycol. Various other materials can be present as coatings or else can modify the physical form of a solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar or the like. A syrup or elixir may contain the polypeptide of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form must be pharmaceutically acceptable and substantially non-toxic in the amounts employed. The polypeptides of the invention may also be incorporated into sustained release preparations and devices.

  Preparations and formulations for oral administration can also be provided with enteric coatings that allow the constructs of the invention to be resistant to the gastric environment and to pass into the intestine. More generally, preparations and formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. A suitable suppository may also be used for delivery into the gastrointestinal tract.

  The polypeptides of the present invention may also be administered intravenously or intraperitoneally by infusion or injection. Specific examples are as further described in pages 144 and 145 of WO 08/020079.

  For topical administration, the polypeptides of the invention may be applied in pure form (ie when they are liquid). In general, however, it will be desirable to administer them to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which can be a solid or liquid. Specific examples are as further described on page 145 of WO 08/020079.

  In general, the concentration of the polypeptide of the present invention in a liquid composition (such as a lotion) can be from about 0.1 to 25 wt-%, preferably from about 0.5 to 10 wt-%. The concentration in a semi-solid or solid composition (such as a gel or powder) can be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

  The amount of the polypeptide of the invention required for use in treatment is not only the specific polypeptide selected, but also the route of administration, the nature of the condition being treated, and the age and It may vary with the condition, but will ultimately be at the discretion of the attending physician or clinician. Also, the dosage of the polypeptide of the present invention varies depending on the target cell, tumor, tissue, graft or organ.

  The desired dose may conveniently be presented as a single dose or as divided doses administered at appropriate intervals (eg, as 2, 3, 4 or more sub-doses / day). The sub-dose itself may be further divided, for example, during a number of separate loosely spaced administrations.

  Dosage regimens can include prolonged daily treatments. By "long term" is meant a duration of at least 2 weeks and preferably several weeks, months or years. Necessary modifications in this dosage range can be determined by one of ordinary skill in the art using only routine experimentation with the teachings provided herein. See Remington's Pharmaceutical Sciences (Martin, E.W., ed.4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in any complication event.

  In another aspect, the present invention relates to a method for the prevention and / or treatment of at least one disease and disorder associated with CXCR7, said method comprising providing a subject in need thereof a polypeptide and / or Or administering a pharmaceutically active amount of a pharmaceutical composition comprising the same.

  In the context of the present invention, the term “prevention and / or treatment” not only includes preventing and / or treating the disease, but also generally preventing the onset of the disease, progression of the disease. Delaying or reversing, preventing or delaying the occurrence of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, and / or Any reduction caused by the disease, reducing the severity and / or duration of any symptoms associated therewith and / or preventing further increases in the severity of the disease and / or any symptoms associated therewith Prevention, reduction, or reversal of the physiological damage of and as well as any pharmacological effects that are generally beneficial to the patient being treated.

  The subject to be treated can be any warm-blooded animal, but especially a mammal, more particularly a human. As will be apparent to those skilled in the art, the subject to be treated may in particular be a person suffering from or at risk of the diseases and disorders referred to herein.

  The present invention relates to a method for the prevention and / or treatment of at least one disease or disorder associated with CXCR7, with its biological or pharmaceutical activity and / or a biological pathway comprising CXCR7 Or with signal transduction, the method can be used to provide a subject in need thereof a pharmaceutical composition of an amino acid sequence of the invention, a polypeptide of the invention, a polypeptide of the invention, and / or a pharmaceutical composition comprising the same. Administration of an active amount. In one embodiment, the present invention provides at least one that can be treated by modulating CXCR7, its biological or pharmacological activity, and / or a biological pathway or signaling that includes CXCR7. With regard to a method for the prevention and / or treatment of a disease or disorder, said method provides to a subject in need thereof a pharmaceutically active amount of a polypeptide of the invention and / or a pharmaceutical composition comprising the same. Administration. In one embodiment, the pharmaceutically effective amount is sufficient to modulate CXCR7, its biological or pharmacological activity, and / or a biological pathway or signaling that includes CXCR7. And / or CXCR7, its biological or pharmacological activity, and / or the biological pathways or signaling involving CXCR7 that are sufficient to regulate the circulation of the invention It can be an amount that provides a level of the polypeptide.

  In one embodiment, the invention provides prevention and / or treatment by administering to a patient a polypeptide of the invention, or a nucleotide construct of the invention encoding the same, and / or a pharmaceutical composition comprising the same. It relates to a method for the prevention and / or treatment of at least one disease or disorder which can be made. In one embodiment, the method comprises subjecting a subject in need thereof to a pharmaceutically active amount of a polypeptide of the invention, or a nucleotide construct of the invention encoding the same, and / or a pharmaceutical composition comprising the same. Administration.

  In one embodiment, the invention inhibits binding of CXCL12 and / or CXCL11 to CXCR7 in specific cells or in specific tissues of the subject being treated (and in particular to CXCR7). Prevention and / or prevention of at least one disease or disorder that can be prevented and / or treated by inhibiting CXCL12 and / or CXCL11 binding in cancer cells or in a tumor present in the subject being treated With regard to a method for treatment, said method requires a pharmaceutically active amount of a polypeptide of the invention, or a nucleotide construct of the invention encoding the same, and / or a pharmaceutical composition comprising the same. Administration to a subject.

  In one embodiment, the present invention is a method for the prevention and / or treatment of at least one disease or disorder selected from the group consisting of the diseases and disorders listed herein, said method comprising Administration to a subject in need of a polypeptide of the invention, or a nucleotide construct of the invention encoding the same, and / or a pharmaceutical composition comprising the same.

  In one embodiment, the present invention relates to a method for immunotherapy, in particular passive immunotherapy, which comprises subjecting a subject suffering from or at risk for the diseases and disorders referred to herein to Administering a pharmaceutically active amount of a polypeptide, or a nucleotide construct of the invention encoding the same, and / or a pharmaceutical composition comprising the same.

  In the above methods, the amino acid sequences, polypeptides and / or compositions comprising the same of the present invention can be administered in any suitable manner, depending on the particular pharmaceutical formulation or composition used. . Thus, a polypeptide of the invention and / or a composition comprising the same can be administered, for example, orally, intraperitoneally (eg, intravenous, subcutaneous, muscle, depending on the particular pharmaceutical formulation or composition used). Or via any other route of administration that avoids the gastrointestinal tract), intranasally, transdermally, topically, by suppository. The clinician will determine the appropriate route of administration and suitable pharmaceutical formulation or composition used in such administration, depending on the disease or disorder to be prevented or treated and other factors well known to the clinician, You will be able to choose.

  The polypeptides of the invention and / or compositions comprising the same are administered according to a treatment regimen suitable for preventing and / or treating the disease or disorder to be prevented or treated. The clinician generally chooses a suitable treatment regimen from factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and / or the severity of the symptoms thereof, Can be determined depending on the peptide, the particular route of administration to be used and the pharmaceutical formulation or composition, the age, sex, weight, diet, general condition of the patient, and similar factors well known to the clinician, etc. Will.

  In general, a treatment regimen comprises administration of one or more polypeptides of the invention, or one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. May be included. The particular amount or dose to be administered can be determined again by the clinician based on the factors cited above.

  In general, for the prevention and / or treatment of the diseases and disorders referred to herein, the particular disease or disorder to be treated, the efficacy of the particular polypeptide of the invention used, the particular route of administration, And depending on the particular pharmaceutical formulation or composition used, the polypeptides of the invention will generally be from 1 gram to 0.01 microgram / kg body weight / day, preferably from 0.1 gram to 0 grams. .1 microgram / kg body weight / day, eg, in an amount of about 1, 10, 100, or 1000 microgram / kg body weight / day, continuous (eg, by infusion), as a single daily dose or daily Can be administered as multiple divided doses. The clinician will generally be able to determine a suitable daily dose based on the factors referred to herein. It will also be apparent that in certain cases, the clinician may choose to deviate from these quantities, for example, based on the factors cited above and his expert judgment. In general, some guidance on the amount to be administered, but taking into account differences in affinity / binding power, efficacy, biodistribution, half-life, and similar factors well known to those skilled in the art From the doses normally administered for equivalent conventional antibodies or antibody fragments to the same target administered via the same route.

  In one embodiment, a single contiguous polypeptide of the invention may be used. In one embodiment, two or more polypeptides of the invention are provided in combination.

  The polypeptides of the invention may be used in combination with one or more additional pharmaceutically active compounds or ingredients, ie as a combination treatment regimen, which may or may not lead to a synergistic effect. . Again, the clinician will be able to select such additional compounds or components, as well as a suitable combination of treatment regimens, based on the factors cited above and his expert judgment.

  In particular, the polypeptides of the invention are used in combination with other pharmaceutically active compounds or ingredients that can be used or used for the prevention and / or treatment of the diseases and disorders cited herein. As a result, a synergistic effect may or may not be obtained. Examples of such compounds and components, and routes, methods, and pharmaceutical formulations or compositions for their administration may be apparent to the clinician, but generally, for treatment of the tumor to be treated. Therefore, it contains an active ingredient which is usually applied to suppress cell growth.

  Specific contemplated combinations for use with the polypeptides of the invention for oncology include, but are not limited to, eg, CXCR4 antagonists such as, for example, AMD3100, other chemokine receptors Body antagonists, taxol, etc .; gemcitobin; cisplatin; cIAP inhibitors (eg, inhibitors to cIAP1, cIAP2, and / or XIAP); MEK inhibitors, including but not limited to, eg, U0126, PD0325901 A bRaf inhibitor, but includes but is not limited to, eg, RAF265; and an mTOR inhibitor, but includes but is not limited to, eg, RAD001; a VEGF inhibitor, but includes but is not limited to, eg, bevacizumab, sunitinib ( sutinib) and Sorafu Nibs; Her 2 inhibitors, including but not limited to, eg, trastuzumab and lapatinib; PDGFR, FGFR, src, JAK, STAT, and / or GSK3 inhibitors; selective estrogen receptor modulators, but not limited to Including estrogen receptor down-regulators, but not limited to fulvestrant. Specific contemplated combinations for use with the polypeptides of the invention for inflammatory conditions include, but are not limited to, for example, interferon beta 1 alpha and beta, natalizumab; TNF alpha antagonists (but not limited to Including, for example, infliximab, adalimumab, certolizumab pegol, etanercept); disease modifying anti-rheumatic drugs such as methotrexate (MTX); glucocorticoids (but including but not limited to hydrocortisone); Steroidal anti-inflammatory drugs (including but not limited to, for example, ibuprofen, sulindac).

  Other specific compounds / polypeptides that can be used in combination with the compounds / polypeptides of the invention are amino acid sequences and polypeptides directed against CXCR4, which are international applications by Ablynx NV WO 09/138519, non-prepublished US application 61 / 358,495 by Ablynx NV (filed 25 June 2010); PCT application PCT / EP210 / 064766 by Ablynx NV (filed 4 October 2010); and / Or as described in Ablynx NV PCT application PCT / EP2011 / 050156 (filed Jan. 7, 2011).

  When two or more substances or components are used as part of a combination treatment regimen, they are either substantially at the same time or at different times (e.g. via the same route of administration or via different routes of administration). , Substantially simultaneously, sequentially, or according to alternative regimens). When substances or components are administered simultaneously via the same route of administration, they may be administered as part of different pharmaceutical formulations or compositions or combined pharmaceutical formulations or compositions and will be apparent to those skilled in the art. That's right.

  Also, when two or more active substances or components are used as part of a combination treatment regimen, each of the substances or components is in accordance with the same regimen in the same amount used when the compound or component is used on its own. It may be administered and such combined use may or may not lead to a synergistic effect. However, if the combined use of two or more active substances or ingredients leads to a synergistic effect, the amount of one, more or all of the substances or ingredients to be administered is reduced and still achieve the desired therapeutic effect It may also be possible. This avoids, limits, or reduces any unwanted side effects associated with, for example, the use of one or more of the substance or ingredient (which is still desirable when used in their normal amounts). To obtain a medicinal or therapeutic effect).

  The effectiveness of the treatment regimen used in accordance with the present invention may be determined and / or followed in any manner known per se for the disease or disorder involved, as will be apparent to the clinician. The clinician can also change or modify specific treatment regimens as appropriate and on a case-by-case basis to achieve the desired therapeutic effect and avoid, limit, or reduce unwanted side effects. And / or on the one hand achieving the desired therapeutic effect and on the other hand achieving a suitable balance that avoids, limits or reduces unwanted side effects.

  In general, a treatment regimen can be followed until the desired therapeutic effect is achieved and / or as long as the desired therapeutic effect is maintained. Again, this can be determined by the clinician.

  In another aspect, the present invention is for the prevention and / or treatment of at least one disease and disorder associated with CXCR7; and / or for use in one or more of the methods of treatment referred to herein. To the use of the polypeptides of the invention in the preparation of a pharmaceutical composition.

  The subject to be treated can be any warm-blooded animal, but especially a mammal, more particularly a human. In veterinary applications, subjects to be treated include any animal produced for commercial purposes or kept as a pet. As will be apparent to those skilled in the art, the subject to be treated may in particular be a person who suffers from or is at risk for the diseases and disorders referred to herein.

  The present invention relates to at least one disease or disorder that can be prevented and / or treated by administering a polypeptide of the present invention, or a nucleotide encoding the same, and / or the same pharmaceutical composition to a patient. It relates to the use of a polypeptide of the invention, or a nucleotide encoding the same, in the preparation of a pharmaceutical composition for prevention and / or treatment.

  More particularly, the present invention relates to pharmaceutical compositions for the prevention and / or treatment of diseases and disorders associated with CXCR7, in particular for the prevention and treatment of one or more of the diseases and disorders listed herein. It relates to the use of a polypeptide of the invention, or a nucleotide encoding the same, in the preparation of a product.

  Again, in such pharmaceutical compositions, one or more polypeptides of the invention, or nucleotides encoding the same, and / or the same pharmaceutical composition, and also one or more other activities. It may be combined appropriately with ingredients (such as those mentioned herein).

  The invention also relates to in vitro (eg in vitro or cellular assays) or in vivo (eg in single-cell or multicellular organisms, in particular in mammals, more particularly in humans, for example for the diseases or disorders of the invention). It relates to a composition (eg, a pharmaceutical composition or preparation as further described herein, without limitation) for use either in at-risk or suffering humans.

  In the context of the present invention, “modulating” or “to modulate” means to reduce or inhibit the activity of CXCR7 and in particular CXCR7 (SEQ ID NO: 1), suitable in vitro, cellular or in vivo assays (E.g., as referred to herein) as measured. In particular, the activity of CXCR7 and especially CXCR7 (SEQ ID NO: 1) is compared to the activity of CXCR7 and especially CXCR7 (SEQ ID NO: 1) in the same assay under the same conditions (but without the presence of the polypeptide of the invention). Reduced or inhibited by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, such as at least 50%, at least 60%, at least 70%, at least 80%, or more than 90%. Is as determined using a suitable in vitro, cellular, or in vivo assay, such as those referred to herein.

  Modulation includes, for example, reducing or inhibiting the binding of CXCR7 to one of its substrates or ligands and / or competing with a natural ligand (CXCL11 and / or CXCL12), a substrate for binding to CXCR7. sell.

1.4. Production of Polypeptides of the Invention The present invention further relates to methods for preparing or producing the single variable domains, polypeptides, nucleic acids, host cells, products, and compositions of the immunoglobulins described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

In general, these methods may include the following steps:
a) providing a set, collection, or library of immunoglobulin single variable domains; and b) capable of binding to and / or affinity for CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). Screening said set, collection, or library of immunoglobulin single variable domains for immunoglobulin single variable domains having; and c) capable of binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) And / or isolating amino acid sequences having affinity therefor.

  In such methods, the set, collection, or library of immunoglobulin single variable domains can be any suitable set, collection, or library of immunoglobulin single variable domains. For example, a single variable domain set, collection, or library of immunoglobulins is a set, collection, or library of immunoglobulin sequences (as described herein), eg, a naive set, collection of immunoglobulin sequences, Or a library; a synthetic or semi-synthetic set, collection, or library of immunoglobulin sequences; and / or a set, collection, or library of immunoglobulin sequences that are subjected to affinity maturation.

  Also, in such methods, a set, collection, or library of immunoglobulin single variable domains is a set, collection, or library of heavy or light chain variable domains (such as VL, VH, or VHH domains). It can be. For example, a single variable domain set, collection, or library of immunoglobulins can be a domain antibody or a set, collection, or library of single domain antibodies, or function as a domain antibody or single domain antibody. It can be a set, collection, or library of immunoglobulin single variable domains.

  In a preferred aspect of this method, the set, collection or library of immunoglobulin single variable domains is suitable, for example, using or based on or derived from CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) An immune set, collection of immunoglobulin sequences derived from a mammal that has been appropriately immunized with an antigenic determinant (eg, an antigenic portion, fragment, region, domain, loop, or other epitope thereof), Or it can be a library. In one particular aspect, the antigenic determinant can be an extracellular portion, region, domain, loop, or other extracellular epitope.

  In the above methods, a single variable domain set, collection, or library of immunoglobulins may be displayed on phage, phagemids, ribosomes, or suitable microorganisms (eg, yeast) to facilitate screening. Suitable methods, techniques, and host organisms for displaying and screening immunoglobulin single variable domains (sets, collections, or libraries) can be determined by those of skill in the art, for example, based on further disclosure herein. It will be clear. A reference is also made in the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, a method for generating a single variable domain of an immunoglobulin comprises at least the following steps:
a) providing a collection or sample of cells expressing a single variable domain of an immunoglobulin;
b) screening said collection or sample of cells for cells expressing an amino acid sequence capable of binding to and / or having affinity for CXCR7 and in particular human CXCR7 (SEQ ID NO: 1); and c) i) isolating the amino acid sequence; or ii) isolating a nucleic acid sequence encoding the amino acid sequence from the cell and subsequently expressing the amino acid sequence.

In another aspect, a method for generating an amino acid sequence directed against CXCR7 and particularly human CXCR7 (SEQ ID NO: 1) may comprise at least the following steps:
a) providing a set, collection, or library of nucleic acid sequences that encode a single variable domain of an immunoglobulin;
b) screening said set, collection, or library of nucleic acid sequences for nucleic acid sequences that can bind to and / or have affinity for CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) And c) isolating the nucleic acid sequence, followed by expressing the amino acid sequence.

  In such methods, a set, collection, or library of nucleic acid sequences that encode a single variable domain of an immunoglobulin includes, for example, a naive set, collection, or set of nucleic acid sequences that encode an immunoglobulin sequence, Collection, or library; a set, collection, or library of nucleic acid sequences that encode a synthetic or semi-synthetic set, collection, or library of immunoglobulin sequences; and / or an immunoglobulin sequence that has been subjected to affinity maturation A set, collection or library of nucleic acid sequences encoding the set, collection or library.

In another aspect, a method for generating an amino acid sequence directed against CXCR7 and particularly human CXCR7 (SEQ ID NO: 1) may comprise at least the following steps:
a) providing a set, collection, or library of nucleic acid sequences that encode a single variable domain of an immunoglobulin;
b) encoding an amino acid sequence capable of binding to and / or having affinity for CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) and cross-blocked or single variable of the immunoglobulins of the invention Screening said set, collection, or library of nucleic acid sequences for nucleic acid sequences that cross-block domains or polypeptides (eg, SEQ ID NOs: 39-43, 91, or 99-102 (Table B-3)); And c) isolating the nucleic acid sequence, followed by expressing the amino acid sequence.

  The present invention also determines by the above method, or alternatively by a method comprising one of the above methods, and also at least the nucleotide or amino acid sequence of said immunoglobulin sequence; and It is obtained by a process of expressing or synthesizing said amino acid sequence in a manner known per se (for example by expression in a suitable host cell or host organism or by chemical synthesis).

  Also, following the above steps, one or more immunoglobulin single variable domains of the invention may be appropriately humanized, camelized, or otherwise sequence optimized (eg, manufacturability, stability, and (Or sequences optimized for solubility); and / or the amino acid sequences thus obtained can be combined with each other or into one or more other suitable immunoglobulin single variable domains ( Optionally, they may be linked (via one or more suitable linkers). In addition, the nucleic acid sequence encoding the amino acid sequence of the present invention is appropriately humanized, camelized, or otherwise sequence optimized (eg, a sequence optimized for manufacturability, stability, and / or solubility). And / or one or more nucleic acid sequences encoding the amino acid sequences of the invention into one or more nucleic acid sequences encoding each other or other suitable immunoglobulin single variable domains ( Optionally (via a nucleotide sequence encoding one or more suitable linkers) may be linked, after which the nucleotide sequence obtained in this way may be appropriately expressed, A polypeptide is provided.

  The invention further includes the application and use of the single variable domains, compounds, constructs, polypeptides, nucleic acids, host cells, products, and compositions of the immunoglobulins described herein, as well as CXCR7 and in particular human CXCR7 (sequence It relates to a method for diagnosis, prevention and / or treatment for diseases and disorders related to number 1). Some preferred but non-limiting applications and uses will become clear from the further description herein.

  The present invention also relates to immunoglobulin single variable domains, compounds, constructs, polypeptides, nucleic acids, host cells, products, and compositions described herein for use in therapy.

  In particular, the invention also prevents by administering to a subject in need thereof an amino acid sequence, compound, construct, or polypeptide (a pharmaceutically effective amount thereof) as described herein. Or immunoglobulin single variable domains, compounds, constructs, polypeptides, nucleic acids, host cells, products, and compositions described herein for use in the treatment of a disease or disorder that can be treated.

  More particularly, the present invention relates to immunoglobulin single variable domains, compounds, constructs, polypeptides, nucleic acids, host cells, products, and compositions described herein for use in the treatment of cancer.

1.5. Polypeptides of the present invention and variants of single variable domains of immunoglobulins Polypeptides of the present invention and single variable domains of immunoglobulins, which form part of the polypeptides of the present invention, are potent or otherwise desired. Changes may be made to further improve the properties.

In general, a single variable domain of an immunoglobulin has the formula 1:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 can be defined as a polypeptide with
Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and here, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively.

  Some particularly preferred, but non-limiting combinations of CDR sequences, and preferred combinations of CDR and framework sequences are noted in Table B-2, which is a number of preferred (but (Non-limiting) lists the CDR and framework sequences present in a single variable domain of an immunoglobulin. As will be apparent to those skilled in the art, combinations of CDR1, CDR2, and CDR3 sequences that occur in the same clone (ie, CDR1, CDR2, and CDR3 sequences referred to in the same row or column in Table B-2) are usually (However, the present invention is not limited thereto in its broadest sense and includes other suitable combinations of CDR sequences referred to in Table B-2). Also, a combination of CDR and framework sequences that occur in the same clone (ie, CDR and framework sequences referred to in the same row or column in Table B-2) will usually be preferred (although the present invention). Are not limited to them in their broadest sense, and are other suitable combinations of CDR sequences and framework sequences referred to in Table B-2, as well as such CDR sequences and other suitable frameworks. Including combinations of sequences, eg, as further described herein).

Also, in a single variable domain of an immunoglobulin of the invention comprising a combination of CDRs referred to in Table B-2, each CDR is at least 80%, preferably at least 90%, more preferably a CDR referred to Can be replaced by a CDR selected from the group consisting of a single variable domain of an immunoglobulin having at least 95%, even more preferably at least 99% sequence identity (as defined herein); :
i) any amino acid substitution in such CDR is preferably a conservative amino acid substitution (as defined herein) compared to the corresponding CDR sequence referred to in Table B-2; And / or ii) any such CDR sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions compared to the corresponding CDR sequences referred to in Table B-2; and / or Or iii) any such CDR sequence is a CDR derived using techniques known per se for affinity maturation, in particular starting from the corresponding CDR sequences mentioned in Table B-2 .

  However, as will be apparent to those skilled in the art, the CDR sequences (combinations) and the CDR and framework sequences (combinations) referred to in Table B-2 will generally be preferred.

  Thus, in the immunoglobulin single variable domain of the invention, at least one of the CDR1, CDR2, and CDR3 sequences present is a group consisting of the CDR1, CDR2, and CDR3 sequences listed in Table B-2, respectively. Or; each of at least one of the CDR1, CDR2, and CDR3 sequences listed in Table B-2, respectively, and at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least From the group of CDR1, CDR2, and CDR3 sequences having 99% “sequence identity” (as defined herein); and / or CDR1, CDR2, and CDR3, respectively, listed in Table B-2 At least one of the sequences and 3, 2, or just 1 It is suitably selected from the group consisting of CDR1, CDR2 and CDR3 sequences having “mino acid differences” (as defined herein).

In this context, by appropriate selection, where applicable, the CDR1 sequence is selected from the appropriate CDR1 sequences (ie, as defined herein) and the CDR2 sequence is the appropriate CDR2 sequence (ie, the specification). Each means that the CDR3 sequence is selected from a suitable CDR3 sequence (ie as defined herein). More particularly, the CDR sequence preferably represents the Nanobody of the invention as having an affinity (EC50 value or alternatively an IC ₅₀ value) as defined herein for CXCR7 and in particular human CXCR7 (SEQ ID NO: 1). Are selected to bind with various in vitro and / or in vivo potency or as appropriately measured and / or expressed in other assays, as further described herein.

  In particular, in the immunoglobulin single variable domain of the invention, at least the CDR3 sequence present is from the group consisting of the CDR3 sequences listed in Table B-2, or at least one of the CDR3 sequences listed in Table B-2 From the group consisting of CDR3 sequences having a sequence identity of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%; and / or CDR3 as listed in Table B-2 Suitably selected from the group consisting of CDR3 sequences having at least one of the sequences and a difference of 3, 2, or only 1 amino acid.

  Preferably, in the immunoglobulin single variable domain of the present invention, at least two of the CDR1, CDR2 and CDR3 sequences present are each from the group consisting of the CDR1, CDR2 and CDR3 sequences listed in Table B-2. Or at least one of the CDR1, CDR2, and CDR3 sequences listed in Table B-2, respectively, and at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99, respectively. From the group consisting of CDR1, CDR2, and CDR3 sequences having% sequence identity; and / or, respectively, at least one of the CDR1, CDR2, and CDR3 sequences listed in Table B-2, respectively, 3, 2, Or CDR1 with only one “amino acid difference” , CDR2 and CDR3 sequences.

  In particular, in the immunoglobulin single variable domain of the invention, at least the CDR3 sequence present is from the group consisting of the CDR3 sequences listed in Table B-2, or each of the CDR3 sequences listed in Table B-2. Suitably selected from the group of CDR3 sequences having one and at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity; and CDR1 and CDR2 present At least one of the sequences is at least 80% each from the group of CDR1 and CDR2 sequences listed in Table B-2, respectively, or at least one of the CDR1 and CDR2 sequences listed in Table B-2, respectively. Preferably at least 90%, more preferably at least 95% More preferably from the group of CDR1 and CDR2 sequences having at least 99% sequence identity; and / or, respectively, at least one of the CDR1 and CDR2 sequences listed in Table B-2, respectively 3, 2, or Appropriately selected from the group consisting of CDR1 and CDR2 sequences with only one amino acid difference.

  Most preferably, in the immunoglobulin single variable domain of the invention, each from the group consisting of the CDR1, CDR2, and CDR3 sequences listed in Table B-2, or each listed in Table B-2. CDR1, CDR2, and CDR3 having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2, and CDR3 sequences, respectively. From the group of sequences; and / or, respectively, CDR1, CDR2, having at least one amino acid difference of 3, 2, or only 1 amino acid with at least one of the CDR1, CDR2, and CDR3 sequences listed in Table B-2, respectively. And a CDR3 sequence.

  Even more preferably, at least one of the CDR1, CDR2, and CDR3 sequences present in the single variable domain of an immunoglobulin of the invention consists of the CDR1, CDR2, and CDR3 sequences listed in Table B-2, respectively. Appropriately selected from the group. Preferably, in this aspect, at least one or preferably both of the other two CDR sequences present are at least 80%, preferably at least one of the corresponding CDR sequences listed in Table B-2, respectively. From CDR sequences having at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity; and / or at least one of the corresponding sequences listed in Table B-2, respectively, Each is suitably selected from the group consisting of CDR sequences having 3, 2 or only 1 amino acid difference.

  In particular, at least the CDR3 sequence present in the single variable domain of the immunoglobulin of the present invention is suitably selected from the group consisting of CDR3 listed in Table B-2. Preferably, in this aspect, at least one and preferably both of the CDR1 and CDR2 sequences present are each at least 80%, preferably at least 90%, respectively, with the CDR1 and CDR2 sequences listed in Table B-2. More preferably from a group of CDR1 and CDR2 sequences having at least 95%, even more preferably at least 99% sequence identity; and / or at least one of the CDR1 and CDR2 sequences listed in Table B-2, respectively. And is suitably selected from the group consisting of CDR1 and CDR2 sequences with 3, 2, or only 1 amino acid difference, respectively.

  Even more preferably, at least two of the CDR1, CDR2 and CDR3 sequences present in the single variable domain of the immunoglobulins of the invention consist of the CDR1, CDR2 and CDR3 sequences listed in Table B-2, respectively. Appropriately selected from the group. Preferably, in this aspect, the remaining CDR sequences present are at least 80%, preferably at least 90%, more preferably at least 95% with at least one of the corresponding CDR sequences listed in Table B-2 More preferably from a group of CDR sequences having at least 99% sequence identity; and / or at least one of the corresponding CDR sequences listed in Table B-2 and 3, 2, or only 1 amino acid difference Is suitably selected from the group consisting of CDR sequences having

  In particular, in the immunoglobulin single variable domain of the present invention, at least the CDR3 sequence is appropriately selected from the group consisting of the CDR3 sequences listed in Table B-2, and either the CDR1 sequence or the CDR2 sequence is a It is appropriately selected from the group consisting of CDR1 sequence or CDR2 sequence listed in B-2. Preferably, in this aspect, the remaining CDR sequences present are at least 80%, preferably at least 90%, more preferably at least 95% with at least one of the corresponding CDR sequences listed in Table B-2 More preferably from a group of CDR sequences having at least 99% sequence identity; and / or a CDR having a difference of 3, 2, or only 1 amino acid with the corresponding CDR sequence listed in Table B-2 Appropriately selected from the group consisting of sequences.

  Even more preferably, all three CDR1, CDR2, and CDR3 sequences present in the single variable domain of an immunoglobulin of the invention consist of the CDR1, CDR2, and CDR3 sequences listed in Table B-2, respectively. Appropriately selected from the group.

  Also, in general, combinations of CDRs listed in Table B-2 (ie, those mentioned in the same row or column in Table B-2) are preferred. Thus, a CDR in a single variable domain of an immunoglobulin of the invention is a CDR sequence referred to in Table B-2, or at least 80% with a CDR sequence listed in Table B-2, Preferably from a group of CDR sequences having at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity; and / or the CDR sequences listed in Table B-2 and 3, 2 Or at least one of the other CDRs and preferably both are CDR sequences belonging to the same combination in Table B-2 (ie, when selected from the group consisting of CDR sequences having only one amino acid difference) At least 80%, preferably less, with CDR sequences that are more appropriately selected (refer to the same row or column in Table B-2) or belong to the same combination CDR sequences belonging to the group of CDR sequences having 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) and / or belonging to the same combination It is generally preferred to be appropriately selected from the group of CDR sequences having 3, 2 or only 1 amino acid difference. The other priorities shown in the above paragraph also apply to the CDR combinations mentioned in Table B-2.

  Thus, using non-limiting examples, a polypeptide of the invention can be, for example, a CDR1 sequence having greater than 80% sequence identity with one of the CDR1 sequences referred to in Table B-2, Table B- CDR2 sequences having one, two, or one amino acid difference from one of the CDR2 sequences referred to in 2 (but belonging to different combinations) and CDR3 sequences may be included.

  Some preferred immunoglobulin single variable domains of the invention can include, for example: (1) a CDR1 sequence having greater than 80% sequence identity with one of the CDR1 sequences referred to in Table B-2 One of the CDR2 sequences mentioned in Table B-2 (but belonging to different combinations) and a CDR2 sequence having a difference of 3, 2, or 1 amino acid; and mentioned in Table B-2 (but A CDR3 sequence having greater than 80% sequence identity with one of the CDR3 sequences belonging to a different combination; or (2) greater than 80% sequence identity with one of the CDR1 sequences referred to in Table B-2 CDR1 sequence having; CDR2 sequence and one of the CDR3 sequences listed in Table B-2; or (3) CDR1 sequence; one and more than 80% of the CDR2 sequences listed in Table B-2 A CDR2 sequence having identity; and a CDR3 sequence having 3, 2, or 1 amino acid difference from the CDR3 sequence referred to in Table B-2, belonging to the same combination as the CDR2 sequence.

  Some particularly preferred immunoglobulin single variable domains of the invention may include, for example: (1) CDR1 having greater than 80% sequence identity with one of the CDR1 sequences referred to in Table B-2 A CDR2 sequence having 3, 2, or 1 amino acid difference from the CDR2 sequence mentioned in Table B-2 belonging to the same combination; and a CDR3 sequence mentioned in Table B-2 belonging to the same combination CDR3 sequences having sequence identity greater than 80%; (2) CDR1 sequences; CDR2 listed in Table B-2 and CDR3 sequences listed in Table B-2 (where the CDR2 and CDR3 sequences are different Can belong to a combination).

  Some even more preferred immunoglobulin single variable domains of the invention may include, for example: (1) having greater than 80% sequence identity with one of the CDR1 sequences referred to in Table B-2 CDR1 sequences; CDR2 sequences listed in Table B-2 belonging to the same combination; and CDR3 sequences referred to in Table B-2 belonging to different combinations; or (2) referred to in Table B-2 CDR1 sequences; CDR2 sequences that differ by 3, 2, or 1 amino acid from the CDR2 sequences mentioned in Table B-2 belonging to the same combination; and listed in Table B-2 belonging to the same or different combinations A CDR3 sequence having more than 80% sequence identity with a CDR3 sequence.

  Particularly preferred immunoglobulin single variable domains of the invention are, for example, CDR1 sequences mentioned in Table B-2, more than 80% sequence identity with the CDR2 sequences mentioned in Table B-2 belonging to the same combination. And CDR3 sequences referred to in Table B-2 belonging to the same combination.

  In the most preferred immunoglobulin single variable domains of the invention, the CDR1, CDR2, and CDR3 sequences present are more suitable than one of the combinations of CDR1, CDR2, and CDR3 sequences listed in Table B-2, respectively. Chosen.

  In another preferred but non-limiting aspect of the invention, (a) CDR1 is between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, eg 5, Have a length such as 6 or 7 amino acid residues; and / or (b) CDR2 is between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, eg 16 And / or (c) CDR3 is between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as 6-23. Have a length such as between amino acid residues.

  In another preferred but non-limiting aspect, the invention provides that the CDR sequences (as defined herein) are SEQ ID NOs: 39-43 or 91 and 99-102 (see Table B-3). A) greater than 80%, more preferably greater than 90%, preferably greater than 95%, eg 99% or more sequence identity with at least one CDR sequence of an immunoglobulin single variable domain As defined in).

Another preferred but non-limiting aspect of the present invention is the humanized variant of an immunoglobulin single variable domain of SEQ ID NOs: 39-43 or 91 and 99-102 (see Table B-3). In that it compares at least one humanized substitution (as defined herein), and in particular at least one humanized substitution, as compared to the corresponding native V _HH sequence, with its framework sequence (defined herein). At least one).

  A single variable domain of an immunoglobulin referred to herein as “preferred” (or “more preferred” or “even more preferred”, etc.) is also present in the polypeptides described herein. It will be apparent to those skilled in the art that it is preferred (or more preferred or even more preferred) for use. Thus, a polypeptide comprising or consisting essentially of a single variable domain of one or more “preferred” immunoglobulins of the present invention is generally preferred and one or more “more preferred” of the present invention. A polypeptide comprising or consisting essentially of a single variable domain of an immunoglobulin is generally more preferred, etc.

1.6. Nucleotide of the invention, host cell Another aspect of the invention relates to a nucleic acid encoding an amino acid sequence of the invention (eg, a single variable domain of an immunoglobulin of the invention) or a polypeptide of the invention comprising the same. Again, as generally described herein, such a nucleic acid can be in the form of a gene construct as defined herein. Specific embodiments of this aspect of the invention are provided in Table B-6, SEQ ID NOs: 59-63 and 73-77.

  In another preferred but non-limiting aspect, the invention relates to a single variable domain nucleic acid sequence of an immunoglobulin, wherein the sequence (as defined herein) is SEQ ID NO: 59-63. Or at least one of the nucleic acid sequences of immunoglobulin single variable domains from 73 to 77 (see Table B-6) and greater than 80%, preferably greater than 90%, more preferably greater than 95%. Have greater than, for example, 99% or more sequence identity (as defined herein).

  In another aspect, the invention relates to a nucleic acid sequence comprising a single variable domain nucleic acid sequence of an immunoglobulin, wherein the sequence (as defined herein) is SEQ ID NO: 59-63 or 73-77 ( At least one sequence of an immunoglobulin single variable domain of Table B-6) and more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequences. Have identity (as defined herein).

  In another aspect, the invention expresses or is capable of expressing an amino acid sequence of the invention (eg, a single variable domain of an immunoglobulin, etc.) and / or a polypeptide of the invention comprising the same; and / or Alternatively, it relates to a host or host cell containing the nucleic acid of the present invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

As will be apparent to those skilled in the art, one particularly useful method for preparing the polypeptides of the invention generally comprises the following steps:
i) a nucleic acid (also referred to herein) in a suitable host cell or host organism (also referred to herein as a “host of the present invention”) or encoding said amino acid sequence, polypeptide of the present invention. Expression in another suitable expression system) (referred to as “the nucleic acid of the invention”) in
In some cases, the following continues:
ii) isolating and / or purifying the polypeptide of the present invention thus obtained.
In particular, such a method may comprise the following steps:
i) culturing and / or maintaining the host of the invention under conditions in which the host of the invention expresses and / or produces at least one polypeptide of the invention; optionally followed by:
ii) isolating and / or purifying the polypeptide of the present invention thus obtained.

  The nucleic acid of the present invention may be in the form of single- or double-stranded DNA or RNA, but is preferably in the form of double-stranded DNA. For example, the nucleotide sequences of the present invention can be genomic DNA, cDNA, or synthetic DNA, such as DNA with codon usage specifically adapted for expression in the intended host cell or host organism.

  In accordance with one aspect of the invention, the nucleic acids of the invention are in substantially isolated form, as defined herein.

  The nucleic acids of the invention can also be in the form of a vector (eg, a plasmid, cosmid, or YAC, etc.), can be present in and / or be part thereof, It can be in isolated form.

The nucleic acids of the invention can be prepared or obtained in a manner known per se and / or suitable based on the information about the single variable domains of immunoglobulins about the polypeptides of the invention given herein. Isolated from natural sources. To provide analogs, nucleotide sequences encoding naturally occurring V _HH domains can be subjected to, for example, site-directed mutagenesis, and nucleic acids of the invention encoding the analogs are provided. Also, as will be apparent to those skilled in the art, to prepare the nucleic acids of the present invention, and also several nucleotide sequences, such as at least one nucleotide sequence encoding a polypeptide of the present invention and such as one or more Nucleic acids encoding these linkers can be linked together in a suitable manner.

  Techniques for producing the nucleic acids of the invention will be apparent to those of skill in the art, but include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; two or more naturally occurring and / or synthetic Combining sequences (or two or more parts thereof), introducing mutations that lead to expression of the cleaved expression product; introducing one or more restriction sites (eg, easy digestion and / or using suitable restriction enzymes) Or making cassettes and / or regions that can be ligated), and / or using one or more “mismatch” primers, eg, naturally occurring sequences of CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) Introducing mutations using a PCR reaction using as a template. These and other techniques will be apparent to those skilled in the art, but reference will again be made to standard handbooks referred to above, such as Sambrook et al. And Ausubel et al., And the examples below. Made.

  The nucleic acids of the invention can also be in the form of a genetic construct, can be present in and / or be part thereof, as will be apparent to those skilled in the art, and WO 08/020079 As described on pages 131-134 (incorporated herein by reference). Such gene constructs are generally typically one or more elements of a gene construct known per se, such as one or more suitable regulatory elements (eg suitable promoters, enhancers, terminators, etc. And at least one nucleic acid of the invention linked to additional elements of the genetic construct referred to herein. Such a gene construct comprising at least one nucleic acid of the present invention may also be referred to herein as a “gene construct of the present invention”.

  The gene construct of the present invention can be DNA or RNA, but is preferably double-stranded DNA. The gene construct of the present invention is also in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or host organism, or It can be in a form suitable for independent replication, maintenance, and / or inheritance in the intended host organism. For example, the gene construct of the present invention may be in the form of a vector such as a plasmid, cosmid, YAC, viral vector, or transposon. In particular, the vector can be an expression vector, ie, a vector that can provide expression in vitro and / or in vivo (eg, in a suitable host cell, host organism, and / or expression system).

In preferred but non-limiting aspects, the genetic constructs of the invention include: i) at least one nucleic acid of the invention; operably connected to ii) one or more regulatory elements, Such as a promoter and optionally a suitable terminator; and, optionally,
iii) one or more additional elements of a gene construct known per se;
Here, the terms “operably connected” and “operably linked” have the meaning given in pages 131 to 134 of WO 08/020079; and where “regulatory element”, “Promoter”, “terminator”, and “further element” are as described on pages 131-134 of WO 08/020079; and where the gene construct is pages 131-134 of WO 08/020079. As described further below.

  The nucleic acid of the invention and / or the gene construct of the invention may be used to transform a host cell or host organism, ie, for the expression and / or production of a polypeptide of the invention. Suitable hosts or host cells will be apparent to those skilled in the art, e.g. any suitable fungus, prokaryotic or eukaryotic cell or cell line or any suitable fungus, prokaryotic or eukaryotic organism, e.g. As described on pages 134 and 135 of WO 08/020079; and per se for expression and production of antibodies and antibody fragments, including but not limited to (single) domain antibodies and ScFv fragments Can be any other known host or host cell, but will be apparent to those skilled in the art. References are also made to the general background art cited hereinabove, as well as, for example, WO 94/29457; WO 96/34103; WO 99/42077.

  The single variable domains and polypeptides of the immunoglobulins of the present invention can also be found, for example, in the milk of a transgenic animal, such as in the milk of a rabbit, cow, goat, or sheep (eg, transgene in a mammal). See US-A-6,741,957, US-A-6,304,489, and US-A-6,849,992 for general techniques for introducing In plant parts (including but not limited to their leaves, flowers, fruits, seeds, roots or tubers) (for example in tobacco, corn, soy or alfalfa) or, for example, silkworm Bombix mori It can also be produced in cocoons.

  Furthermore, the immunoglobulin single variable domains and polypeptides of the invention can also be expressed and / or produced in a cell-free expression system, suitable examples of such systems being apparent to those skilled in the art. Let's go. Some preferred but non-limiting examples include expression in a wheat germ system; in a rabbit reticulocyte lysate; or in an E. coli Zubay system.

  As mentioned above, one of the advantages of using an immunoglobulin single variable domain is that a polypeptide based thereon can be prepared through expression in a suitable bacterial system, and suitable bacterial expression systems, vectors The host cells, regulatory elements, etc. will be apparent to those skilled in the art, for example, from the references cited above. However, it should be noted that the present invention, in its broadest sense, is not limited to expression in bacterial systems.

  Preferably, the present invention uses an expression system (such as a bacterial expression system) that provides the polypeptide of the invention in a form suitable for pharmaceutical use (in vivo or in vitro), such expression system. Will again be apparent to those skilled in the art. Also, as will be apparent to those skilled in the art, the polypeptides of the present invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.

  For industrial scale production, preferred heterologous hosts for (industrial) production of protein therapeutics comprising immunoglobulin single variable domains or immunoglobulin single variable domains are those of large scale expression / production / fermentation. And strains of E. coli, Pichia pastoris, S. cerevisiae that are particularly suitable for large scale pharmaceutical (ie GMP grade) expression / production / fermentation. Suitable examples of such strains will be apparent to those skilled in the art. Such strains and production / expression systems are also made available by companies such as Richter Helm (Hamburg, Germany) or CMC Biologics (Soeborg, Denmark).

  Alternatively, mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, can be used for large-scale expression / production / fermentation and especially for large-scale pharmaceutical expression / production / fermentation. Again, such expression / production systems are also made available by some of the companies mentioned above.

  The choice of a particular expression system may depend, in part, on the requirements for specific post-translational modifications, more specifically glycosylation. Production of recombinant proteins comprising a single variable domain of an immunoglobulin where glycosylation is desirable or required may require the use of a mammalian expression host that has the ability to glycosylate the expressed protein. In this regard, it will be apparent to one skilled in the art that the resulting glycosylation pattern (ie, the nature of the saccharide, the number and position of the attached residues) may depend on the cell or cell line used for expression. I will. Preferably, either human cells or cell lines are used (ie, leading to a protein having a substantially human glycosylation pattern) or another mammalian cell is used, which Glycosylation patterns that are substantially and / or functionally the same or at least mimic human glycosylation can be provided. In general, prokaryotic hosts (eg, E. coli, etc.) do not have the ability to glycosylate proteins, and the use of lower eukaryotes (eg, yeast) is usually different from human glycosylation. Lead to the pattern. Nevertheless, it should be understood that all preceding host cells and expression systems can be used in the present invention depending on the desired polypeptide to be obtained.

  Thus, according to one non-limiting aspect of the invention, the polypeptides of the invention are glycosylated. According to another non-limiting aspect of the invention, the polypeptides of the invention are non-glycosylated.

  In accordance with one preferred but non-limiting aspect of the present invention, the polypeptides of the present invention can be used in bacterial cells, particularly bacterial cells suitable for large-scale pharmaceutical production (eg, the strains referred to above). Cell).

  In accordance with another preferred but non-limiting aspect of the present invention, the polypeptides of the present invention can be used in yeast cells, particularly yeast cells suitable for large-scale pharmaceutical production (eg, the species referred to above). Cell).

  In accordance with yet another preferred but non-limiting aspect of the present invention, the polypeptide of the present invention is expressed in mammalian cells, in particular in human cells or cells of human cell lines, more particularly in humans. Produced in cells or cells of a human cell line suitable for large scale pharmaceutical production, such as the cell lines referred to herein above.

  The immunoglobulins of the invention when expression in a host cell is used to produce a single variable domain of an immunoglobulin, and a polypeptide of the invention, as further described on pages 138 and 139 of WO 08/020079 Single variable domains and polypeptides of (eg, in the cytosol, in the periplasm, or in inclusion bodies) and then isolated from the host cell and optionally further purified. Or it can be produced extracellularly (eg, in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. Thus, according to one non-limiting aspect of the present invention, the polypeptides of the present invention are produced extracellularly and isolated from host cells, in particular from bacterial cells, or from inclusion bodies in bacterial cells. Amino acid sequence and polypeptide. In accordance with another non-limiting aspect of the present invention, the amino acid sequence or polypeptide of the present invention is an amino acid sequence or polypeptide that is produced extracellularly and isolated from the culture medium in which the host cells are cultured.

  Some preferred but non-limiting promoters for use with these host cells include those mentioned on pages 139 and 140 of WO 08/020079.

  Some preferred but non-limiting secretory sequences for use with these host cells include those mentioned in pages 139 and 140 of WO 08/020079.

  Suitable techniques for transforming a host or host cell of the present invention will be apparent to those skilled in the art and may depend on the intended host cell / host organism and the gene construct used. References are again made to the handbooks and patent applications mentioned above.

  Following transformation, steps may be performed to detect and select those host cells or host organisms that have been successfully transformed with the nucleotide sequences / gene constructs of the present invention. This can be, for example, a selection step based on a selectable marker present in the gene construct of the invention or a step comprising detection of the amino acid sequence of the invention (eg using a specific antibody).

  A transformed host cell (which can be in the form of a stable cell line) or host organism (which can be in the form of a stable mutant strain or strain) forms a further aspect of the invention. Preferably, these host cells or host organisms express or express the polypeptides of the invention (in the case of host organisms: in at least one cell, part, tissue or organ thereof). Be (at least) possible (eg under suitable conditions). The invention also includes further generations, progeny, and / or progeny of the host cells or host organisms of the invention that can be obtained, for example, by cell division or by sexual or asexual reproduction.

  In order to produce / obtain expression of a single variable domain of an immunoglobulin of the invention, the transformed host cell or transformed host organism is generally kept and maintained under conditions and / or It may be cultured to express / produce the (desired) amino acid sequence or polypeptide of the invention. Suitable conditions will be apparent to those skilled in the art, but will usually depend on the host cell / host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequences of the invention. Again, references are made to the handbooks and patent applications mentioned above in the paragraph relating to the genetic constructs of the present invention.

  In general, suitable conditions include the use of a suitable medium, the presence of a suitable source of food and / or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducer or compound. (Eg, when the nucleotide sequence of the invention is under the control of an inducible promoter); all of which can be selected by one skilled in the art. Again, under such conditions, the single variable domains of the immunoglobulins of the invention can be expressed in a constitutive manner, in a transient manner, or only when appropriately induced.

  The amino acid sequences or polypeptides of the invention may also be (initially) produced in immature form (as mentioned above), which in turn depends on the host cell / host organism used. It will be apparent to those skilled in the art that post-translational modifications may be made. The amino acid sequences or polypeptides of the invention may also be glycosylated again, depending on the host cell / host organism used.

  The amino acid sequences or polypeptides of the invention can then be obtained from the host cell / host organism and / or from the culture medium in which the host cell or host organism has been cultured, per se known protein isolation and / or purification techniques, for example ( Preparative) chromatography and / or electrophoresis techniques, differential precipitation techniques, affinity techniques (eg, using specific cleavable amino acid sequences fused to the amino acid sequences or polypeptides of the invention), and / or preparation May be isolated using conventional immunological techniques (ie, using an antibody against the amino acid sequence to be isolated).

  The entire contents of all references (including literature and references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby incorporated by reference, particularly in this specification. The teachings referenced above in the book are expressly incorporated.

1.7 Modulators of CXCR7 Utilizing a number of different screening protocols, agents that modulate the level of CXCR7 activity or function in cells, particularly in mammalian cells, and particularly in human cells. Can be identified. In general terms, screening methods include, for example, binding to CXCR7 or a fragment thereof and the polypeptide or ISVD of the invention (eg, SEQ ID NOs: 39-48, 78-89, 91, 99-102, or Prevent the binding of CXCR7 (SEQ ID NO: 1) to CXCR7 (SEQ ID NO: 1), and interact with (human) CXCR7 (SEQ ID NO: 1) Identifying one or more agents. In some embodiments, the agent exhibits at least about 1.5, 2, 3, 4, 5, 10, 20, 50, 100, 300, 500, or 1000 times the affinity of the agent for another protein. It binds to CXCR7. In some embodiments, only a few, eg, 300, 250, 200, fragments of CXCR7 contain the epitopes described herein (and optionally contain additional non-CXCR7 amino acids at the N and / or C terminus). , 150, 100, 50, 40, 30, 20 or less amino acids. In some embodiments, a CXCR7 fragment is any fragment having fewer than all amino acids in a full length CXCR7 polypeptide.

  In some embodiments, CXCR7 modulators are identified by screening for molecules that compete with a polypeptide of the invention or ISVD from binding to a CXCR7 polypeptide or fragment thereof. Those skilled in the art will recognize that there are numerous ways to perform competitive analysis, such as those disclosed herein. In some embodiments, a sample with CXCR7 is treated with a labeled polypeptide or ISVD of the invention (eg, any one of SEQ ID NOs: 39-48, 78-89, 91, 99-102, or 132-140). Contain) and then contact with potential competitor molecules. A change (eg, decrease) in the amount of polypeptide or ISVD bound to CXCR7 in the presence of the test compound indicates that the test compound is a potential CXCR7 modulator.

1.8 Kits for use in diagnostic, research, and therapeutic applications suggested on kits for use in diagnostic and / or prognostic applications are also provided by the present invention. For diagnostic and research applications, such kits may include any or all of the following: assay reagents, buffers, and anti-CXCR7 polypeptides or ISVDs of the invention. The therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.

  The kit can also include instructional materials that include instructions (ie, protocols) for carrying out the methods of the invention. Indicator materials typically include written or printed materials, but they are not limited to such. Any medium capable of storing such instructions and communicating them to the end user is contemplated by the present invention. Such media include, but are not limited to, electronic storage media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional material.

  The invention is further described herein using the following non-limiting preferred aspects, figures, and examples.

Preferred non-limiting aspects:
Aspect A-1: A single variable domain of an immunoglobulin directed to and / or capable of specifically binding to CXCR7 and a specific human CXCR7 (SEQ ID NO: 1).

  Aspect A-2: Single variable domain of an immunoglobulin according to aspect A-1, in substantially isolated form.

  Aspect A-3: A single variable domain of an immunoglobulin according to aspect A-1 or A-2 for administration to a subject, wherein said single variable domain of said immunoglobulin is said subject Does not occur naturally.

Aspect A-4: CXCR7 and especially human CXCR7 (SEQ ID NO: 1) have a dissociation constant (KD) of 10 ⁻⁵ to 10 ⁻¹² mol / liter or less, and preferably 10 ⁻⁷ to 10 ⁻¹² mol / liter or less, And, more preferably, a single variable domain of an immunoglobulin that can specifically bind using 10 ⁻⁸ to 10 ⁻¹² mol / liter. Such an immunoglobulin single variable domain may in particular be an immunoglobulin single variable domain according to any of the preceding aspects.

Aspects A-5: in CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), during the association rate _{(K on} rate) 10 ² M ^-1 s ^-1 ~ about 10 ⁷ M ^-1 s ^-1, preferably 10 ³ M ⁻¹ s ⁻¹ to 10 ⁷ M ⁻¹ s ⁻¹ , more preferably between 10 ⁴ M ⁻¹ s ⁻¹ to 10 ⁷ M ⁻¹ s ⁻¹ , for example, 10 ⁵ M ⁻¹ s ⁻¹ to A single variable domain of an immunoglobulin that can specifically bind, such as between 10 ⁷ M ⁻¹ s ⁻¹ . Such an immunoglobulin single variable domain may in particular be an immunoglobulin single variable domain according to any of the preceding aspects.

Aspects A-6: in CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), between the dissociation rate _{(K off} rate) 1s ^{-1 ~10 -6} s ^-1, preferably ^{10 -2} s ^{-1 ~10 -6} s ^-1 , more preferably between 10 ⁻³ s ⁻¹ to 10 ⁻⁶ s ⁻¹ , for example between 10 ⁻⁴ s ⁻¹ to 10 ⁻⁶ s ⁻¹ , etc. A single variable domain of immunoglobulin that can. Such an immunoglobulin single variable domain may in particular be an immunoglobulin single variable domain according to any of the preceding aspects.

  Aspect A-7: an immunoglobulin capable of specifically binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Single variable domain. Such an immunoglobulin single variable domain may in particular be an immunoglobulin single variable domain according to any of the preceding aspects.

  Aspect A-8: SDF-1 and / or I-TAC (CXCL11 and / or CXCL12) on CXCR7 and especially human CXCR7 (SEQ ID NO: 1) less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example Of an immunoglobulin that can be specifically substituted with an average KI of less than 1 nM and an average SDF-1 and / or I-TAC substitution of 50% or more, more preferably 75% or more, and even more preferably 80% or more. A single variable domain. Such average Ki and / or average displacement value may be determined, for example, in an assay as described in Example 9 or 10.

  Aspect A-9: SDF-1 and / or I-TAC (CXCL11 and / or CXCL12) on CXCR7 and especially human CXCR7 (SEQ ID NO: 1), an average KI of less than 20 nM and an average SDF-1 of 70% or more A single variable domain of an immunoglobulin that can be specifically substituted with / or I-TAC substitution. Such average Ki and / or average displacement value may be determined, for example, in an assay as described in Example 9 or 10.

  Aspect A-10: Single variable of immunoglobulin according to any of the preceding aspects, consisting essentially of four framework regions (respectively FR1 to FR4) and three complementarity determining regions (respectively CDR1 to CDR3) domain.

  Aspect A-11: A single variable domain of an immunoglobulin according to any of the preceding aspects, which is an immunoglobulin sequence.

  Aspect A-12: Single variable domain of an immunoglobulin according to any of the preceding aspects, which is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence.

  Aspect A-13: An immunoglobulin sequence according to any of the preceding aspects, which is a humanized immunoglobulin sequence, camelized immunoglobulin sequence, or an immunoglobulin sequence obtained by techniques such as affinity maturation. One variable domain.

  Aspect A-14: Single immunoglobulin according to any of the preceding aspects consisting essentially of a light chain variable domain sequence (eg, VL sequence); or consisting of a heavy chain variable domain sequence (eg, VH sequence) Variable domain.

  Aspect A-15: According to any of the preceding aspects consisting essentially of a heavy chain variable domain sequence derived from a conventional four chain antibody or consisting essentially of a heavy chain variable domain sequence derived from a heavy chain antibody A single variable domain of an immunoglobulin.

Aspect A-16: Domain antibody (or immunoglobulin sequence suitable for use as a domain antibody), single domain antibody (or immunoglobulin sequence suitable for use as a single domain antibody), “ of an immunoglobulin according to any of the preceding aspects, which may be a "dAb" (or an immunoglobulin sequence suitable for use as a dAb), or a Nanobody, including but not limited to a _VHH sequence. A single variable domain.

  Aspect A-17: A single variable domain of an immunoglobulin according to any of the preceding aspects, consisting essentially of Nanobodies.

Aspect A-18: A single variable domain of an immunoglobulin according to any of the preceding aspects, consisting essentially of a Nanobody that is:
i) at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOS: 1-22 of WO 2009/138519, for the purpose of determining the degree of amino acid identity Ignoring the amino acid residues that form the CDR sequences;
And where:
ii) Preferably, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering are holes referred to in Table A-1. Selected from mark residues.

Aspect A-19: An immunoglobulin single variable domain according to any of the preceding aspects, consisting essentially of an immunoglobulin single variable domain that is: i) SEQ ID NOs: 39-43, 91, or 99- At least 80% amino acid identity with at least one of the 102 immunoglobulin single variable domains, wherein the amino acid residues forming the CDR sequences are ignored for purposes of determining the degree of amino acid identity ;;
And where:
ii) Preferably, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering are holes referred to in Table A-1. Selected from mark residues.

Aspect A-20: A single variable domain of an immunoglobulin according to any of the preceding aspects consisting essentially of a polypeptide comprising: i) an immunoglobulin having SEQ ID NO: 39-43, 91, or 99-102 A single variable domain of a first immunoglobulin having at least 80% amino acid identity with a single variable domain of an immunoglobulin selected from the group of single variable domains of wherein a degree of amino acid identity is determined For purposes, ignore the amino acid residues that form the CDR sequences;
And it consists of:
ii) a single variable domain of a second immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin having SEQ ID NO: 2, where for purposes of determining the degree of amino acid identity Ignoring the amino acid residues that form the CDR sequences; and optionally including: iii) a linker.

  Aspect A-21: A single variable domain of an immunoglobulin according to any of the previous aspects, consisting essentially of a single variable domain of a humanized or otherwise sequence optimized immunoglobulin.

  Aspect A-22: In addition to at least one binding site for binding to CXCR7 and particularly human CXCR7 (SEQ ID NO: 1), one or more additional binding sites for binding to other antigens, proteins, or targets A single variable domain of an immunoglobulin according to any of the preceding aspects, comprising:

CDR-based aspects Aspect B-1: Amino acid residues directed to and / or capable of specifically binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) and selected from the group consisting of: An immunoglobulin single variable domain comprising one or more (preferably one) stretch of:
a) a single variable domain of an immunoglobulin of SEQ ID NO: 9-13, 93 or 107-110;
b) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 9-13, 93 or 107-110;
c) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 9-13, 93 or 107-110;
d) a single variable domain of an immunoglobulin of SEQ ID NO: 19-23, 95 or 115-118;
e) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 19-23, 95 or 115-118;
f) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 19-23, 95 or 115-118;
g) a single variable domain of an immunoglobulin of SEQ ID NO: 29-33, 97 or 123-126;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 29-33, 97 or 123-126;
i) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 29-33, 97 or 123-126;
Or any suitable combination thereof.
Such an immunoglobulin single variable domain may in particular be a VHH or a sequence-optimized V _HH (such as humanized, stabilized and / or solubilized VHH).

  Aspect B-2: A single variable domain of an immunoglobulin according to aspect B-1, wherein at least one of said stretches of amino acid residues is bound to CXCR7 and in particular to human CXCR7 (SEQ ID NO: 1) Part of the antigen binding site for

Aspect B-3: two or more amino acid residues directed against and / or capable of specifically binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), and selected from the group consisting of A single variable domain of an immunoglobulin containing a stretch of:
a) a single variable domain of an immunoglobulin of SEQ ID NO: 9-13, 93 or 107-110;
b) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 9-13, 93 or 107-110;
c) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 9-13, 93 or 107-110;
d) a single variable domain of an immunoglobulin of SEQ ID NO: 19-23, 95 or 115-118;
e) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 19-23, 95 or 115-118;
f) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 19-23, 95 or 115-118;
g) a single variable domain of an immunoglobulin of SEQ ID NO: 29-33, 97 or 123-126;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 29-33, 97 or 123-126;
i) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 29-33, 97 or 123-126;
(I) if the first stretch of amino acid residues corresponds to one of the single variable domains of an immunoglobulin according to a), b), or c), the second stretch of amino acid residues is d) , E), f), g), h), or i) to correspond to one of the single variable domains of an immunoglobulin; (ii) a first stretch of amino acid residues is d) , E), or f) when corresponding to one of the immunoglobulin single variable domains, the second stretch of amino acid residues is a), b), c), g), h), or correspond to one of the single variable domains of the immunoglobulin according to i); (iii) the first stretch of amino acid residues is g), h), or the immunoglobulin single according to i) If it corresponds to one of the variable domains, the second string of amino acid residues Ji is, a), b), c), d), e), or made to correspond to one of the single variable domain of an immunoglobulin according to f).
Such immunoglobulin single variable domains can in particular be VHH or sequence optimized VHH (eg humanized, stabilized and / or solubilized VHH, etc.).

  Aspect B-4: A single variable domain of an immunoglobulin according to aspect B-3, wherein at least two stretches of amino acid residues are for binding to CXCR7 and in particular to human CXCR7 (SEQ ID NO: 1) Part of the antigen binding site.

Aspect B-5: of an immunoglobulin directed against and / or specifically capable of binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) and comprising three or more stretches of amino acid residues A single variable domain, wherein the first stretch of amino acid residues is selected from the group consisting of:
a) a single variable domain of an immunoglobulin of SEQ ID NO: 9-13, 93 or 107-110;
b) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 9-13, 93 or 107-110;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 9-13, 93 or 107-110; the second of the amino acid residues The stretch is selected from the group consisting of:
d) a single variable domain of an immunoglobulin of SEQ ID NO: 19-23, 95 or 115-118;
e) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 19-23, 95 or 115-118;
f) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 19-23, 95 or 115-118; and The third stretch is selected from the group consisting of:
g) a single variable domain of an immunoglobulin of SEQ ID NO: 29-33, 97 or 123-126;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 29-33, 97 or 123-126;
i) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from at least one of the immunoglobulin single variable domains of SEQ ID NOs: 29-33, 97 or 123-126.
Such immunoglobulin single variable domains can in particular be VHH or sequence optimized VHH (eg humanized, stabilized and / or solubilized VHH, etc.).

  Aspect B-6: A single variable domain of an immunoglobulin according to aspect B-5, wherein at least three stretches of amino acid residues are for binding to CXCR7 and in particular to human CXCR7 (SEQ ID NO: 1) Part of the antigen binding site.

Aspect B-7: A single variable domain of an immunoglobulin directed against and / or capable of specifically binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), wherein said immunity The CDR sequence of the globulin single variable domain is at least 70% amino acid identity, preferably at least 80%, with at least one CDR sequence of the immunoglobulin single variable domain of SEQ ID NOs: 39-43, 91 or 99-102. % Amino acid identity, more preferably at least 90% amino acid identity, such as greater than 95% amino acid identity or even substantially 100% amino acid identity. CDR sequences are preferentially determined via Kabat, as defined herein.
Such immunoglobulin single variable domains can in particular be VHH or sequence optimized VHH (eg humanized, stabilized and / or solubilized VHH, etc.).

  Aspect C-1: immunoglobulin of SEQ ID NO: 39-43, 91, or 99-102 directed against CXCR7 and especially human CXCR7 (SEQ ID NO: 1), and to CXCR7 and especially human CXCR7 (SEQ ID NO: 1) A single variable domain or polypeptide of an immunoglobulin that cross-blocks the binding of at least one of the single variable domains of: Such immunoglobulin single variable domains are in particular the immunoglobulin single according to any of aspects A-1 to A-22 and / or according to any of aspects B-1 to B-7. It can be a variable domain. Also preferably, such an immunoglobulin single variable domain is capable of specifically binding to CXCR7 and particularly human CXCR7 (SEQ ID NO: 1).

  Aspect C-2: immunoglobulin of SEQ ID NO: 39-43, 91, or 99-102 directed against CXCR7 and especially human CXCR7 (SEQ ID NO: 1), and to CXCR7 and especially human CXCR7 (SEQ ID NO: 1) An immunoglobulin single variable domain or polypeptide that is cross-blocked from binding by a polypeptide of at least one of the single variable domains of SEQ ID NOs: 44-48, 78-89, or 131-140 Its fragments). Such immunoglobulin single variable domains are in particular the immunoglobulin single according to any of aspects A-1 to A-22 and / or according to any of aspects B-1 to B-7. It can be a variable domain. Also preferably, such an immunoglobulin single variable domain is capable of specifically binding to CXCR7 and particularly human CXCR7 (SEQ ID NO: 1).

  Aspect C-3: A single variable domain or polypeptide of an immunoglobulin according to either aspect C-1 or C-2, wherein the single variable domain of said immunoglobulin cross-blocks or The ability to cross block is detected in a displacement assay (eg, as described in Examples 9 and / or 10 below).

  Aspect C-4: A single variable domain or polypeptide of an immunoglobulin according to any of aspects C-1 or C-3, wherein said single variable domain of said immunoglobulin cross-blocks or The ability to be cross-blocked is detected in an ELISA assay.

  Aspect D-1: A single variable domain of an immunoglobulin according to any of aspects B-1 to B-7 or C-1 to C-7, which is in substantially isolated form.

  Aspect D-2: A single variable domain of an immunoglobulin according to any of aspects B-1 to B-7, C-1 to C-7, and / or D1 for administration to a subject, Here, the single variable domain of the immunoglobulin does not naturally occur in the subject.

Aspect D-3: CXCR7 and especially human CXCR7 (SEQ ID NO: 1) have a dissociation constant (KD) of 10 ⁻⁵ to 10 ⁻¹² mol / liter or less, and preferably 10 ⁻⁷ to 10 ⁻¹² mol / liter or less, And, more preferably, 10 ⁻⁸ to 10 ⁻¹² mol / liter can be used to specifically bind Aspects B-1 to B-7, C-1 to C-7, and / or D1 to D A single variable domain of an immunoglobulin according to any of -2.

Aspect D-4: CXCR7 and especially human CXCR7 (SEQ ID NO: 1) have a dissociation constant (KD) of 10 ⁻⁵ to 10 ⁻¹² mol / liter or less, and preferably 10 ⁻⁷ to 10 ⁻¹² mol / liter or less, And, more preferably, 10 ⁻⁸ to 10 ⁻¹² mol / liter can be used to specifically bind Aspects B-1 to B-7, C-1 to C-7, and / or D1 to D A single variable domain of an immunoglobulin according to any of -3.

Aspect D-5: in CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), between the dissociation rate _{(K off} rate) 1s ^{-1 ~10 -6} s ^-1, preferably ^{10 -2} s ^{-1 ~10 -6} s ^-1 , more preferably between 10 ⁻³ s ⁻¹ to 10 ⁻⁶ s ⁻¹ , for example between 10 ⁻⁴ s ⁻¹ to 10 ⁻⁶ s ⁻¹ , etc. A single variable domain of an immunoglobulin according to any of aspects B-1 to B-7, C-1 to C-7, and / or D1 to D-4.

  Aspect D-6: Aspect B that can specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. A single variable domain of an immunoglobulin according to any of -1 to B-7, C-1 to C-7, and / or D1 to D-5. The immunoglobulin single variable domain according to aspects D-1 to D-6 may in particular be an immunoglobulin single variable domain according to any of aspects A-1 to A-22.

  Aspect E-1: Aspects B-1 to B-7, C, which are naturally occurring immunoglobulin single variable domains (from any suitable species) or synthetic or semi-synthetic immunoglobulin single variable domains A single variable domain of an immunoglobulin according to any of -1 to C-7 and / or D1 to D-6.

  Aspect E-2: Sequence-optimized immunoglobulin according to any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and / or E-1 A single variable domain.

  Aspect E-3: Stabilized, according to Aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and / or D-1 or D-2 A single variable domain of an immunoglobulin.

  Aspect E-4: Aspects B-1 to B-7, C-1 to C-7, D1, which are naturally occurring immunoglobulin sequences (from any suitable species) or synthetic or semi-synthetic immunoglobulin sequences A single variable domain of an immunoglobulin according to any of ~ D-6 and / or E-1 to E-3.

  Aspect E-5: Aspects B-1 to B-7, C-1 which are humanized immunoglobulin sequences, camelized immunoglobulin sequences, or immunoglobulin sequences obtained by technology (eg, affinity maturation, etc.) A single variable domain of an immunoglobulin according to any of C-7, D1 to D-6, and / or E-1 to E-4.

Aspect E-6: Aspects B-1 to B-7, C-1 consisting essentially of a light chain variable domain sequence (eg, _VL sequence); or consisting essentially of a heavy chain variable domain sequence (eg, _VH sequence) A single variable domain of an immunoglobulin according to any of -C-7, D1-D-6, and / or E-1 -E-5.

  Aspect E-7: Aspects B-1 to B-7 consisting essentially of a heavy chain variable domain sequence derived from a conventional four chain antibody or consisting essentially of a heavy chain variable domain sequence derived from a heavy chain antibody, A single variable domain of an immunoglobulin according to any of C-1 to C-7, D1 to D-6, and / or E-1 to E-6.

Aspect E-8: Domain antibody (or immunoglobulin sequence suitable for use as a domain antibody), single domain antibody (or immunoglobulin sequence suitable for use as a single domain antibody), “ dAb "(or are immunoglobulin sequences that are suitable for use as dAb), or Nanobodies (but not limited _to, a _{V HH} sequence) may be, aspects B-1~B-7, C- 1 A single variable domain of an immunoglobulin according to any of -C-7, D1-D-6, and / or E-1 -E-7.

  Aspect E-9: According to any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and / or E-1 to E-8 consisting essentially of Nanobodies. Single immunoglobulin variable domain.

Aspect E-10: Aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and / or E-1 consisting essentially of a single variable domain of an immunoglobulin that is: Single variable domain of an immunoglobulin according to any of -E-9:
i) having at least 80% amino acid identity with at least one of the immunoglobulin single variable domains described herein, wherein a CDR sequence is formed for the purpose of determining the degree of amino acid identity. Ignore amino acid residues
And where:
ii) Preferably, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering is the hole referred to in Table B-2. Selected from mark residues.

Aspect E-11: Aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and / or E-1 consisting essentially of a single variable domain of an immunoglobulin that is: Single variable domain of an immunoglobulin according to any of ~ E-10:
i) at least 80% amino acid identity with at least one of the immunoglobulin single variable domains of SEQ ID NOs: 39-43, 91, or 99-102, wherein the purpose is to determine the degree of amino acid identity To ignore the amino acid residues that form the CDR sequences;
And where:
ii) Preferably, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 according to Kabat numbering is the hole referred to in Table B-2. Selected from mark residues.

  Aspect E-12: Aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and / or E-1 substantially consisting of a single variable domain of a humanized immunoglobulin A single variable domain of an immunoglobulin according to any of E-8.

  Aspect E-13: In addition to at least one binding site for binding formed by a CDR sequence, includes one or more additional binding sites for binding to other antigens, proteins, or targets A single variable domain of an immunoglobulin according to any of -1 to B-7, C-1 to C-7, D1 to D-6, and / or E-1 to E-8. An immunoglobulin single variable domain according to aspects E-1 to E-13 may in particular be an immunoglobulin single variable domain according to any of aspects A-1 to A-22.

Polypeptide Aspect K-1: Aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, and / or E-1 to E-13 A polypeptide comprising one or more immunoglobulin single variable domains according to any of the above, optionally further comprising one or more peptide linkers.

  Aspect K-2: In addition, a polypeptide according to aspect K-1, comprising a single variable domain of one or more (preferably one) immunoglobulin directed against serum albumin.

  Aspect K-3: Polypeptide according to aspect K-1 or K-2, wherein said immunoglobulin single variable domain directed against serum albumin is directed against human serum albumin.

  Aspect K-4: Aspect K-1 or K- wherein the one or more immunoglobulin single variable domains directed against serum albumin are immunoglobulin single variable domains with SEQ ID NO: 2. 4. A polypeptide according to any of 3.

  Aspect K-5: Any of Aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, and / or E-1 to E-13 A polypeptide comprising one or more immunoglobulin single variable domains, one or more cytotoxic payloads, and optionally further comprising one or more peptide linkers.

  Aspect K-6: Any of Aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, and / or E-1 to E-13 One or more immunoglobulin single variable domains according to the above, one or more (preferably one) immunoglobulin single variable domains (Nanobodies) directed against CXCR4, and optionally In addition, a polypeptide comprising or consisting essentially of one or more peptide linkers.

  Aspect K-7: at least one (preferably one) immunoglobulin single variable domain (preferably a Nanobody) and at least one (cyto) cytotoxic group, moiety directed against (human) CXCR7 Or a polypeptide comprising or consisting essentially of a payload (optionally chemically or linked via one or more suitable linkers or spacers).

  Aspect K-8: at least one (preferably one) immunoglobulin single variable domain (preferably a Nanobody) directed against (human) CXCR7, at least one directed against (human) CXCR4 A (preferably one) immunoglobulin single variable domain (preferably a Nanobody) and at least one (cyto) toxic group, moiety, or payload (optionally chemically or one or more suitable) Linked or substantially consisting of a linker or spacer).

  Aspect K-9: at least one (preferably one) immunoglobulin single variable domain (preferably a Nanobody) directed against (human) CXCR7 and at least one directed against (human) CXCR4 A (preferably one) immunoglobulin single variable domain (preferably a Nanobody) (optionally chemically or linked via one or more suitable linkers or spacers) Polypeptide.

  Aspect K-10: at least one (preferably one) immunoglobulin single variable domain (preferably a Nanobody) directed against (human) CXCR7, at least one directed against (human) CXCR4 A (preferably one) immunoglobulin single variable domain (preferably a Nanobody) and a peptide or immunoglobulin single variable domain (preferably a Nanobody) directed against (human) serum albumin (optionally chemical Or consisting essentially of or consisting of one or more suitable linkers or spacers).

  Aspect K-11: they are the same (optionally chemically or linked via one or more suitable linkers or spacers) (immune) two immunity directed against CXCR7 A polypeptide comprising or consisting essentially of a single variable domain of a globulin (preferably a Nanobody).

  Aspect K-12: Two immunoglobulins directed against (human) CXCR7 that are different from each other (optionally chemically or linked via one or more suitable linkers or spacers) A polypeptide comprising or consisting essentially of a single variable domain (preferably a Nanobody).

  Aspect K-13: two immunoglobulin single variable domains (preferably Nanobodies) directed against (human) CXCR7 and peptides or immunoglobulins directed against (human) serum albumin that are the same A polypeptide comprising or consisting essentially of a single variable domain (preferably a Nanobody), optionally linked chemically or via one or more suitable linkers or spacers.

  Aspect K-14: two immunoglobulin single variable domains (preferably Nanobodies) directed against different (human) CXCR7 and peptides or immunoglobulins directed against (human) serum albumin A polypeptide comprising or consisting essentially of a single variable domain (preferably a Nanobody) (optionally chemically or linked via one or more suitable linkers or spacers).

Nucleic acid Aspect M-1: Any of Aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13 A nucleic acid or nucleotide sequence encoding a polypeptide according to any of the single variable domains of immunoglobulins according to aspect K-1 to K-4.

  Aspect M-2: Nucleic acid or nucleotide sequence with SEQ ID NO: 59-63, 73-77, or 99 (Table B-6).

Host cell Aspect N-1: Any of Aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13 A polypeptide according to any of the single variable domains of aspects according to aspects K-1 to K-4, or can be expressed under suitable circumstances; and / or A host or host cell comprising a nucleic acid or nucleotide sequence according to aspects M-1 to M-2.

Composition Aspect O-1: Any of Aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13 A single variable domain of at least one immunoglobulin according to or at least one polypeptide according to any of aspects K-1 to K-4, or a nucleic acid or nucleotide according to aspects M-1 to M-2 A composition comprising a sequence.

  Aspect O-2: Composition according to aspect O-1, which is a pharmaceutical composition.

  Aspect O-3: A pharmaceutical composition, which further comprises at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and optionally one or more A composition according to aspect O-2 comprising a further pharmaceutically active polypeptide and / or compound.

Production of Drug and Composition of the Present Invention Aspect P-1: Aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 A method for producing an immunoglobulin single variable domain according to any of E-13, or a polypeptide according to any of aspects K-1 to K-4, comprising at least Including the following steps:
a) expressing a nucleic acid or nucleotide sequence according to aspect M-1 or aspect M-2 in a suitable host cell or host organism or in another suitable expression system;
In some cases, the following continues:
b) An immunoglobulin according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13 Isolating and / or purifying a polypeptide according to any of the single variable domains of aspects K-1 to K-4.

Aspect P-2: According to any of the aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13 A method for producing a polypeptide according to any of the single variable domains of immunoglobulins, aspects K-1 to K-4, said method comprising at least the following steps:
a) A host or host cell according to aspect N-1, wherein said host or host cell is selected from aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D- Expressing at least one immunoglobulin single variable domain according to any of 1-D-6, E-1 to E-13, a polypeptide according to any of aspects K-1 to K-4 and / or Or culturing and / or maintaining under conditions to produce;
In some cases, the following continues:
b) An immunoglobulin according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13 Isolating and / or purifying a polypeptide according to any of the single variable domains of aspects K-1 to K-4.

Screening Method Aspect Q-1: Method for screening a single variable domain of an immunoglobulin directed against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) comprising at least the following steps:
a) providing a set, collection, or library of nucleic acid sequences that encode a single variable domain of an immunoglobulin;
b) encodes a single variable domain of an immunoglobulin capable of binding to and / or having affinity for CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), and cross-blocked or Nanobody of the invention (Eg, SEQ ID NOs: 39-43, 91, or 99-102 (Table B-3)), or polypeptides or constructs of the invention (eg, SEQ ID NOs: 44-48, 78-89, or 131-140 (Table See B-4))) screening said set, collection or library of nucleic acid sequences for nucleic acid sequences cross-blocking; and c) isolating said nucleic acid sequences, followed by said immunization To express a single variable domain of globulin.

Use of the Agents of the Invention Aspect R-1: A method for the prevention and / or treatment of cancer and inflammatory diseases (eg, referred to herein), said method comprising a subject in need thereof And at least 1 according to any one of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13. A single variable domain of two immunoglobulins, a polypeptide according to any of aspects K-1 to K-4; or a pharmaceutically active amount of a composition according to aspect O-2 or O-3 Including that.

  Aspect R-2: Biological pathway or signal comprising CXCR7 and especially human CXCR7 (SEQ ID NO: 1), its biological or pharmacological activity, and / or CXCR7 and especially human CXCR7 (SEQ ID NO: 1) A method for the prevention and / or treatment of at least one disease or disorder associated with transmission, wherein said method provides a subject in need thereof with aspects A-1 to A-22, B-1 to B A single variable domain of at least one immunoglobulin according to any of -7, C-1 to C-4, D-1 to D-6, E-1 to E-13, aspects K-1 to K- Administering a pharmaceutically active amount of a polypeptide according to any of 4; or a composition according to aspect O-2 or O-3.

  Aspect R-3: For subjects who need it, Aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 A single variable domain of at least one immunoglobulin according to any of E-13, a polypeptide according to any of aspects K-1 to K-4; or a composition according to aspect O-2 or O-3 A method for the prevention and / or treatment of at least one disease or disorder that can be prevented and / or treated by administering an agent to a subject in need thereof. 1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13. A single variable domain, a polypeptide according to any of aspects K-1 to K-4; Or comprising administering a pharmaceutically active amount of a composition according to the O-3.

  Aspect R-4: A method for immunotherapy, wherein the method provides a subject in need thereof with aspects A-1 to A-22, B-1 to B-7, C-1 to C- 4, at least one immunoglobulin single variable domain according to any of D-1 to D-6, E-1 to E-13, polypeptide according to any of aspects K-1 to K-4 Or administering a pharmaceutically active amount of a composition according to aspect O-2 or O-3.

  Aspect R-5: Aspects A-1 to A-22, B-1 to B-7, C-1 to C for use in one or more of the methods according to aspects R-1 to R-3 -4, a single variable domain of an immunoglobulin according to any of D-1 to D-6, E-1 to E-13, a polypeptide according to any of K-1 to K-4, O- A pharmaceutical composition according to 2 or O-3.

  Aspect R-6: Polypeptide according to any of aspects K-1 to K-4, for diagnosis, prevention, and / or treatment of cancer.

Further aspects:
1. A single variable domain of amino acid residue M33 in CXCR7 (SEQ ID NO: 1) and optionally at least one immunoglobulin that binds to and / or recognizes amino acid residue V32 and / or amino acid residue M37 ( ISVD) and amino acid residue WF19 of CXCR7 (SEQ ID NO: 1) and optionally a construct comprising at least one ISVD that binds to and / or recognizes S23 and / or D25.

  2. The construct according to aspect 1, for use as a medicament for reducing tumor growth and / or treating cancer, preferably head and neck cancer or GBM.

  3. A single immunoglobulin that can specifically substitute SDF-1 and I-TAC on human CXCR7 (SEQ ID NO: 1) with an average Ki of less than 100 nM and an average SDF-1 and I-TAC substitution of 50% or more. One variable domain.

  4). A single variable domain of an immunoglobulin capable of specifically replacing SDF-1 on human CXCR7 (SEQ ID NO: 1) with an average Ki of less than 100 nM and an average SDF-1 substitution of 50% or more.

  5. A single variable domain of an immunoglobulin capable of specifically substituting I-TAC on human CXCR7 (SEQ ID NO: 1) with an average Ki of less than 100 nM and an average I-TAC substitution of 50% or more.

  6). The immunoglobulin single variable domain of any of aspects 3-5, wherein the average Ki is 50 nM or less.

  7). The immunoglobulin single variable domain of any of aspects 3-5, wherein the average Ki is 10 nM or less.

  8). The immunoglobulin single variable domain of any of aspects 3-7, wherein the average SDF-1 or I-TAC substitution is 80% or more.

  9. A single variable domain of an immunoglobulin capable of binding to human CXCR7 (SEQ ID NO: 1) with a Kd of less than 50 nM.

  10. A single variable domain of an immunoglobulin that binds to and / or recognizes amino acid residue M33 in CXCR7 (SEQ ID NO: 1), and optionally amino acid residue V32 and / or amino acid residue M37.

  11. A single variable domain of an immunoglobulin that binds to and / or recognizes amino acid residue WF19 of CXCR7 (SEQ ID NO: 1) and optionally S23 and / or D25.

  12 A single variable domain of an immunoglobulin according to aspect 10 or 11 for use as a medicament for reducing tumor growth and / or treating cancer, preferably head and neck cancer or GBM.

13. A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 9;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 9;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 9;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 19;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 19;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 19;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 29;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 29;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 29.

14 A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 10;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 10;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 10; and wherein CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 20;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 20;
f) an immunoglobulin single variable domain having 3, 2, or 1 amino acid difference from the immunoglobulin single variable domain of SEQ ID NO: 20; and wherein CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 30;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 30;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 30.

15. A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 11;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 11;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 11;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 21;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 21;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 21;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 31;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 31;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 31.

16. A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 12;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 12;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 12;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 22;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 22;
f) a single variable domain of an immunoglobulin having a 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 22;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of an immunoglobulin of SEQ ID NO: 32;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 32;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 32.

17. A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 13;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 13;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 13;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 23;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 23;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 23;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 33;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 33;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 33.

18. A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) the single variable domain of an immunoglobulin of SEQ ID NO: 93;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 93;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 93;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 95;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 95;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 95;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of an immunoglobulin of SEQ ID NO: 97;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 97;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 97.

19. A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 107;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 107;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 107;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 115;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 115;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 115;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 123;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 123;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 123.

20. A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 108;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 108;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 108;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 116;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 116;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 116;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 124;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 124;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 124.

21. A single variable domain of an immunoglobulin of any of aspects 3-12, wherein the single variable domain of the immunoglobulin is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) the single variable domain of the immunoglobulin of SEQ ID NO: 110;
b) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 110;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 110;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 118;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 118;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 118;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 126;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 126;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 126.

  22. A single variable domain of an immunoglobulin according to any of aspects 1-21, wherein the framework region (FR) is SEQ ID NO: 4-8, 92, 103, 104, or 106 (FR1), 14-18, 94, 111, 112, or 114 (FR2), 24-28, 96, 119, 120, or 122 (FR3), and / or 34-38, 98, 127, 128, or 130 (FR4) With more than 80% sequence identity.

  23. A polypeptide comprising a single variable domain of the immunoglobulin of any of aspects 3-22.

  24. A polypeptide according to aspect 23, wherein the immunoglobulin single variable domain is more than 80% identical in sequence to the immunoglobulin single variable domain of SEQ ID NOs: 39-43, 91, or 99-102. Selected from the group consisting of a single variable domain of an immunoglobulin having an amino acid sequence with sex.

  25. In accordance with any of aspects 23-24, in addition, a linker comprising at least one human serum albumin binding immunoglobulin single variable domain, optionally selected from the group of linkers with SEQ ID NOs: 49-58 A polypeptide comprising.

  26. A polypeptide according to any of aspects 23-25, additionally comprising a linker comprising ALB8 (SEQ ID NO: 2) and optionally selected from the group of linkers with SEQ ID NO: 49-58.

  27. A polypeptide according to any of aspects 23-26, wherein the polypeptide has greater than 80% sequence identity with the polypeptides of SEQ ID NOs: 44-48, 78-89, and 131-140. Selected from the group consisting of polypeptides having an amino acid sequence.

28. A construct selected from the group consisting of:
A construct comprising amino acid residue WF19 of CXCR7 (SEQ ID NO: 1) and optionally at least two ISVDs that bind to and / or recognize S23 and / or D25, wherein said at least The two ISVDs can be the same or different;
A construct comprising amino acid residue M33 in CXCR7 (SEQ ID NO: 1) and optionally at least two ISVDs that bind to and / or recognize amino acid residue V32 and / or amino acid residue M37, Where the at least two ISVDs can be the same or different;
A construct comprising at least one group 1 ISVD and at least one group 2 ISVD;
A construct comprising at least one group 1 ISVD and at least one group 3 ISVD;
-A construct comprising at least one group 2 ISVD and at least one group 3 ISVD; and-a construct comprising at least one 01C10-like sequence and at least one 14G03-like sequence.

  29. The construct according to aspect 28, for use as a medicament for reducing tumor growth and / or treating cancer, preferably head and neck cancer or GBM.

30. A nucleic acid sequence encoding: i) a single variable domain of an immunoglobulin according to any of aspects 3-22;
ii) a polypeptide according to any of aspects 23-27; or iii) a construct according to any of aspects 1, 2, 28, or 29.

31. A pharmaceutical composition comprising: i) a single variable domain of an immunoglobulin according to any of aspects 3 to 22;
ii) a polypeptide according to any of aspects 23-27; or iii) a construct according to any of aspects 1, 2, 28, or 29;
And optionally a pharmaceutically acceptable excipient.

  32. A single variable domain of an immunoglobulin according to any of aspects 3 to 22, a polypeptide according to any of aspects 23 to 27, or use in cancer, preferably head and neck cancer, GBM, and / or inflammatory disease A construct according to any of aspects 1, 2, 28, or 29 for

  33. A single variable domain of an immunoglobulin according to any of aspects 3-22, a polypeptide according to any of aspects 23-27, or any of aspects 1, 2, 28, or 29 for use in rheumatoid arthritis Constructs according to

  34. A single variable domain of an immunoglobulin according to any of aspects 3-22, a polypeptide according to any of aspects 23-27, or aspects 1, 2, 28, or 29 for use in multiple sclerosis A construct according to one of the following.

35. Producing a single variable domain of an immunoglobulin according to any of aspects 3-22, a polypeptide according to any of aspects 23-27, or a construct according to any of aspects 1, 2, 28, or 29. A method for performing the method comprising at least the following steps:
a) expressing a nucleic acid or nucleotide sequence according to aspect 30 in a suitable host cell or host organism or in another suitable expression system; optionally followed by:
b) a single variable domain of an immunoglobulin according to any of aspects 3-22, a polypeptide according to any of aspects 23-27, or a construct according to any of aspects 1, 2, 28, or 29. Isolation and / or purification.

36. Formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
A single variable of an immunoglobulin comprising an amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of the immunoglobulin of SEQ ID NO: 9;
Where CDR2 is the immunoglobulin single variable domain of SEQ ID NO: 19; and where CDR3 is the immunoglobulin single variable domain of SEQ ID NO: 29.

37. A single variable of an immunoglobulin comprising an amino acid sequence with formula 1 is represented by formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of the immunoglobulin of SEQ ID NO: 10;
Where CDR2 is the immunoglobulin single variable domain of SEQ ID NO: 20; and where CDR3 is the immunoglobulin single variable domain of SEQ ID NO: 30.

38. A single variable of an immunoglobulin comprising an amino acid sequence with formula 1 is represented by formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of the immunoglobulin of SEQ ID NO: 11;
Where CDR2 is the single variable domain of the immunoglobulin of SEQ ID NO: 21; and where CDR3 is the single variable domain of the immunoglobulin of SEQ ID NO: 31.

39. A single variable of an immunoglobulin comprising an amino acid sequence with formula 1 is represented by formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of the immunoglobulin of SEQ ID NO: 12;
Where CDR2 is the immunoglobulin single variable domain of SEQ ID NO: 22; and where CDR3 is the immunoglobulin single variable domain of SEQ ID NO: 32.

40. A single variable of an immunoglobulin comprising an amino acid sequence with formula 1 is represented by formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of an immunoglobulin of SEQ ID NO: 13;
Where CDR2 is the immunoglobulin single variable domain of SEQ ID NO: 23; and where CDR3 is the immunoglobulin single variable domain of SEQ ID NO: 33.

41. A single variable of an immunoglobulin comprising an amino acid sequence with formula 1 is represented by formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of an immunoglobulin of SEQ ID NO: 93;
Where CDR2 is the immunoglobulin single variable domain of SEQ ID NO: 95; and where CDR3 is the immunoglobulin single variable domain of SEQ ID NO: 97.

42. A single variable of an immunoglobulin comprising an amino acid sequence with formula 1 is represented by formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of an immunoglobulin of SEQ ID NO: 107;
Where CDR2 is the single variable domain of the immunoglobulin of SEQ ID NO: 115; and where CDR3 is the single variable domain of the immunoglobulin of SEQ ID NO: 123.

43. A single variable of an immunoglobulin comprising an amino acid sequence with formula 1 is represented by formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of the immunoglobulin of SEQ ID NO: 108;
Where CDR2 is the single variable domain of the immunoglobulin of SEQ ID NO: 116; and where CDR3 is the single variable domain of the immunoglobulin of SEQ ID NO: 124.

44. A single variable of an immunoglobulin comprising an amino acid sequence with formula 1 is represented by formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refer to framework regions 1-4 and are framework regions of a single variable domain of an immunoglobulin;
Where CDR1 is the single variable domain of an immunoglobulin of SEQ ID NO: 110;
Where CDR2 is the single variable domain of the immunoglobulin of SEQ ID NO: 118; and where CDR3 is the single variable domain of the immunoglobulin of SEQ ID NO: 126.

Experimental part:
Array:

Example 1: Cloning Human CXCR7 (hCXCR7), mouse CXCR7 (Open Biosystems), and cynomolgus monkey (Table B-1) encoding cDNA were cloned into pVAX-1 (Invitrogen) and / or pCDNA 3.1 (Invitrogen) Turned into. Transfection of pVAX1-hCXCR7 and pCDNA3.1 human (mouse) (cynomolgus) CXCR7 constructs in Hek293 cells resulted in CXCR7 cell surface expression, but human CXCR7-specific monoclonal antibody (Mab) 11G8 (R & D Systems) and As shown by FACS analysis using PE-labeled goat anti-mouse IgG (Jackson ImmunoResearch Inc.) to detect the antibody.

Example 2: Immunization For gene immunization, an endotoxin-free pVAX1-CXCR7 plasmid was produced, dissolved at a concentration of 2 mg / ml in 0.9% saline and stored at -20 ° C. Four llamas (391, 395, 396, and 397) were immunized 4 times with 2 mg pVAX1-hCXCR7 via intradermal Jet injection (Akra DermoJet France) with a 2-week interval. . Three weeks after the last DNA immunization, 4 animals had camel kidney (CAKI) cells stably expressing hCXCR7 (Nguyen et al. 2001. Adv. Immunol. 79: 261-296) (2 × 10 ⁷ cells). Booster).

Three llamas (385, 387, and 404) were immunized with two injections of 2 × 10 ⁷ HEK293 transfected with pCDNA3.1-hCXCR7 with an interval of 2 weeks. From llamas 391, 395, 396, and 397, peripheral blood lymphocytes were collected on days 4 and 10 after the last DNA immunization and on days 3 and 9 after cell boost. From llamas 385, 387, and 404, peripheral blood lymphocytes were collected 4 and 8 days after the last cell injection. In addition, biopsy of palpable bow lymph node (LN) was collected from each llama via local surgery on day 3 after the last cell boost. Total RNA was extracted from all lymphocytes with immune tissue and used as a template for preparing cDNA.

Example 3: Library Building The library was built from immune tissues collected from all llamas. Briefly, cDNA was prepared and used from extracted total RNA samples (Example 2) and used as described previously (WO 02/085945 and WO 04/049794) via nested PCR. The repertoire was amplified. The PCR product was digested with SfiI (introduced into the FR1 primer region via nested PCR) and BstEII (a naturally occurring restriction site in FR4), followed by gel electrophoresis, followed by an approximately 400 bp DNA fragment. Purified from gel. The amplified cDNA repertoire was ligated into the corresponding restriction sites of the SfiI-BstEII digested phage display vector (pAX50), resulting in a library after electroporation of E. coli TG1. This display vector allows the production of phage particles and allows individual VHHs (herein also referred to as Nanobodies) to be C-terminal Myc-His6 tags (herein above and also TAG-1). Or expressed as a fusion protein with SEQ ID NO: 71) and with the gene III product.

Recombinant phage that rescues the library by growing the bacteria to log phase (OD ₆₀₀ = 0.5), followed by infection with helper phage, and expressing the cloned Nanobody at the tip of the phage as a pIII fusion protein Got. The phage were stored for further use at 4 ° C. after filter sterilization.

Example 4: Selection of phage displaying human CXCR7-binding Nanobodies Phages from the above library were transformed into hCXCR7 virus-like particles (VLP; Integral Molecular), intact CXCR7-expressing cells, membrane extracts from CXCR7-expressing cells, and Used for selection on peptides.

  In the first selection round, 10 units of VLPs derived from hCXCR7 transfected HEK293 cells were coated on 96 well Maxisorp plates (Nunc) and blocked using low fat milk powder (Marvell 4% in PBS). After 2 hours of incubation with rescued phage, trypsin elution (1 mg / ml) was allowed for 15 minutes at room temperature followed by 20 PBS washes. Protease activity was immediately neutralized by applying 16 mM protease inhibitor ABSF. Round 1 phage output was rescued and a second selection round was performed on 10 or 1 unit of plate-immobilized hCXCR7 VLP. Round 2 phage output selected on 10 or 1 unit plate immobilized hCXCR7 VLPs were infected into TG1 cells and plated on agar plates (LB + Amp + 2% glucose).

  Individual clones of E. coli TG1 infected with the eluted phage pool obtained after selection were picked and grown in 96 deep well plates to produce monoclonal phage after addition of helper phage. Production of monoclonal Nanobodies was induced by the addition of isopropyl-bD-thiogalactopyranoside (IPTG). Periplasmic fractions containing Nanobodies were then prepared by freeze thawing of bacterial pellets in PBS and subsequent centrifugation to remove cell fragments.

Example 5: Identification of CXCR7-specific Nanobodies by Phage ELISA From all round 2 selection outputs, apply a clean, 10-fold dilution of phage supernatant on 2 units of immobilized CXCR7 VLP in phage ELISA And screened. After incubation with HRP-conjugated monoclonal anti-M13 antibody (GE, Cat # 363761) and several washes, phage binding was revealed using TMB substrate (Pierce). The reaction was stopped with H2SO4 and the absorbance was measured using a Sunrise TECAN spectrophotometer (TECAN) at 450 nM. Nanobodies showing an increased ELISA signal at least 2-fold over untransfected control VLPs on hCXCR7 VLPs were considered CXCR7-specific. CXCR7-specific Nanobodies were sequenced and overlapping Nanobodies (identical AA sequences) were removed. This led to the identification of 78 unique sequences belonging to 45 distinct Nanobody B cell lines. Phage ELISA data for representative clones from distinct Nanobody B cell lines is presented in Table B-7, indicating that Nanobodies bind to human CXCR7 on VLPs. Notably, all Nanobodies were derived from PBL after cell boost (except for Nanobody 01C10) (see Example 2). Evaluated against other CXCR7-specific Nanobodies, Nanobody 01C10 was a severely weak binder, which was used in the first example for comparison reasons (data not shown).

Example 6: Identification of CXCR7-specific Nanobodies by FACS analysis Clones representing distinct Nanobody B cell lines were tested as periplasmic extracts for their binding to cell surface exposed CXCR7. In this assay, a 5-fold dilution of the periplasmic extract was incubated with Hek293 hCXCR7 and Hek293wt cells. Nanobody binding was detected using mouse anti-myc (Serotec) followed by anti-mouse IgG-PE (Jackson Immununoresearch). The binding signal (mcf value and binding ratio) of selected Nanobody clones is presented in Table B-8, indicating that Nanobody binds to human CXCR7 of cells.

Example 7: Expression of CXCR7-specific Nanobodies Selected Nanobodies are recloned into the E. coli expression vector pAX100 and expressed as C-terminally linked myc, His6 (hereinafter also Tag-2 or SEQ ID NO: 72) tagged proteins I let you. Various Nanobodies were also expressed as fusion proteins comprising Alb8 (Nanobody-linker-Alb8-myc-His6) (see SEQ ID NOs: 44-48—Table B-4) or as untagged Nanobodies. Expression was induced by IPTG and allowed to continue at 37 ° C for 4 hours. After spinning the cell culture, a periplasmic extract was prepared by freeze thawing the pellet. Nanobodies were purified from these extracts using immobilized metal affinity chromatography (IMAC) and buffer exchange into D-PBS.

Example 8: Binding FACS analysis of CXCR7-specific Nanobodies Serial dilutions of purified protein (concentration range: 400 nM to 180 pM) were incubated with stable HEK-CXCR7 cells for 30 minutes at 4 ° C to allow binding to anti-mouse anti-antibody. Detection was performed using myc (Serotec) and anti-mouse IgG-PE (Jackson Immunoresearch). Half maximum effective concentration (EC50) values and upper plateau levels of selected clones are depicted in Table B-9. These data confirm the screening data and highlight that the indicated Nanobody binds to human CXCR7 of the cells.

  Example 9: Nanobodies compete with SDF-1 for CXCR7 binding (displacement assay).

To assess competitive ability, Nanobodies were evaluated using stable NIH3T3-hCXCR7 cells in an SDF-1 ligand displacement assay. Twenty-four hours after seeding the cells, the cells were incubated at 4 ° C. for 1 hour at a dilution series of purified monovalent Nanobody and the corresponding C-terminal Tag-2 tagged fusion protein to human serum albumin-binding Nanobody Alb8 (Table B- 4: See SEQ ID NOs: 44-48, where all polypeptides are C-terminally tagged with Tag-2). Reference molecules Mab 8F11 (Biolegend), Mab 11G8 (R & D), and unlabeled SDF-1 were also included in the assay. Radiolabeled [ ¹²⁵ I] -CXCL12 was diluted and added to the cells to reach a final concentration of 75 pM. Cells were incubated for 3 hours at 4 ° C. After incubation, the cells were washed twice, lysed using RIPA buffer, and the ¹²⁵ I signal was measured. The average Ki value and the percentage of replacement compared to cold SDF-1 replacement are shown in Table B-10. Competition between Group 1 tested Nanobodies and Mab 8F11 is between 73-83% compared to competition with unlabeled SDF-1. This level of substitution corresponds to 100% blockade of the CXCR7 protein. Because the remaining SDF-1 binding is not considered to be CXCR7 mediated, but is due to SDF-1 interaction with heparin sulfate proteoglycans. Fusion to human serum albumin-bound Nanobody Alb8 has no significant effect on Ki values.

Example 10: Nanobodies compete with SDF-1 for CXCR7 binding (FACS assay)

  The efficacy of Nanobody 07C03 and Mab 8F11 (Biolegend) competing with SDF-1 was evaluated in a competitive FACS using HEK-hCXCR7 cells. Cells were simultaneously incubated at 4 ° C. for 2 hours with 4 nM biotinylated SDF-1 (R & D) and with diluted test molecules. Biotinylated SDF-1 binding was detected using streptavidin PE. The competition curve is depicted in FIG. In this assay, Mab 8F11 and 07C03 competition is complete (> 95%) compared to competition with unlabeled SDF-1, highlighting complete inhibition of the SDF-1-CXCR7 interaction.

Example 11: Epitope mapping The minimal epitope of Mab 11G8 is known to be F14DISSWP20 located at the N-terminus of CXCR7 (see, eg, WO2008 / 048519). Cells were simultaneously incubated at 4 ° C. for 2 hours with 20 nM Mab 11G8 APC (R & D) and with diluted test molecules. The competition curve is depicted in FIG. The level of competition with Mab 11G8 APC ranges from ˜20 to 100%, with each Nanobody epitope to a high degree (high% of competition), to a Mab 11G8 epitope, or to a low degree (low competition) %) Induces allosteric changes that match or affect Mab 11G8 binding. These data indicate that the selected Nanobodies bind to a diverse but possibly overlapping epitope.

  Nanobodies 08A05, 08A10, 07C03, 07B11, 01C10 and 14G03, Mab 8F11 (Biolegend), Mab 11G8 (R & D), and Mab 9C4 (MBL) were further tested in FACS analysis for competition with Alexa647-labeled 14G03. Nanobodies 08A05, 08A10, 07C03, 07B11, Mab 8F11, Mab 11G8, and Mab 9C4 compete with 14G03 binding to CXCR7, and 01C10 does not. It suggests that 01C10 hits an epitope distinct from hits by other selected Nanobodies.

  In the third approach, Nanobodies were tested for their binding to a set of 10 point mutations of CXCR7 (S9A, F14Y, I17L, S18N, W19A, S23G, D25A, V32A, M33Q, N36T) Brought information on the nanobody epitopes. For Nanobodies 08A05, 08A10, 07C03, 07B11, and 14G03, the epitope contained residue M33 but not that of 01C10. The binding of 01C10 (and 07B11) was affected by the W19A mutation, but this mutation did not affect the binding of 08A05, 08A10, 07C03, and 14G03. Again, these data indicate that 01C10 hits a distinct epitope.

Example 12: Mouse / cynomolgus monkey cross-reactivity HEK293 cells transfected with pCDNA3.1-hCXCR7 and pCDNA3.1-mCXCR7, respectively, were used to test cross-reactive binding of Nanobodies to mouse CXCR7 in FACS analysis did. Cells were incubated for 2 hours at 4 ° C. with 32 nM Mab 11G8 (R & D), Mab 9C4 (MBL), Mab 8F11 (Biolegend) or with 800 nM Nanobodies. Nanobody binding was detected using mouse anti-myc (Serotec) and anti-mouse IgG-PE (Jackson Immunoresearch) and Mab binding was detected using goat anti-mouse IgG-PE (Jackson Immunoresearch). Nanobodies 08A10, 14G03, 07B11, and Mab9C4 are not cross-reactive with mouse CXCR7, Nanobodies 08A05 and 07C03 are partially cross-reactive with mouse CXCR7, and Mabs 8F11, Mabs 11G8, and 01C10 are cross-reactive with mouse CXCR7 (Table B-11).

  Cross-reactive binding to cynomolgus monkey CXCR7 was evaluated in the same manner. Nanobodies 08A10, 14G03, 07B11, 08A05, 07C03, 01C10 and Mab 9C4, Mab 8F11, and Mab 11G8 are all cross-reactive with cynomolgus CXCR7 (Table B-11).

Example 13: Building Bivalent and Trivalent Nanobodies Bivalent nanobodies were converted to one N-terminal CXCR7 specific building block (either 01C10, 14G03, 08A05, 08A10, or 07C03, but also less potent) Building with no building blocks (08C02, 01C07, 01D04, etc., they were not listed in the above examples) and C-terminal human serum albumin (HSA) specific building blocks (ALB8) Provide Nanobodies with extended half-life. The trivalent Nanobody consisted of another CXCR7-specific building block to improve the efficacy and effectiveness of Nanobody to displace SDF-1 from the receptor. Bivalent and trivalent Nanobodies were expressed in Pichia with Tag-2 extension.

Example 14: Competition of bivalent and trivalent Nanobodies with SDF-1 binding to CXCR7 Bivalent and trivalent Nanobodies were screened in an SDF-1 displacement assay as described in Example 9. Samples were incubated in the presence or absence of HSA and the effect of HSA binding to Nanobodies was estimated during the assay. Although the potency of divalent nanobodies has dropped dramatically in the presence of HSA, they are much better conserved for trivalent nanobodies (Table B-12).

Example 15: Inhibition of β-arrestin mobilization of bivalent and trivalent Nanobodies
The PathHunter eXpress β-arrestin assay (DiscoverX) was used to evaluate the antagonistic effect of trivalent Nanobodies on β-arrestin mobilization. A panel of 37 trivalent Nanobodies (clones) was screened in the assay at a concentration of 100 nM. The results are ranked in Table B-13 based on the efficiency of inhibition. The most efficient trivalent molecule constitutes a combination with 01C10 (a Nanobody that hits a distinct epitope). (See Example 11). These Nanobodies (clones) can bind to one CXCR7 monomer in a dual manner.

Based on previous results, Nanobodies can be divided into three groups:
-Hits an epitope represented by group 1: 01C10 and apparently distinct from group 2;
-Group 2: represented by 14G03, 08A05, 08A10, and 07C03, apparently hitting an epitope distinct from Group 1; and-Group 3: represented by 07B11, apparently intermediate between Group 1 and Group It is.

  Group 2 (and Group 3) nanobodies, either monovalent or bivalent, demonstrate superior binding and competitive characteristics over the corresponding Group 1 Nanobodies, but in combination with Group 2 Nanobodies Nanobodies gave totally unexpected best results in the β-arrestin mobilization assay.

Example 16: Optimization of Divalent and Trivalent Nanobodies Selected bivalent and trivalent Nanobodies were further characterized and evaluated for efficacy in a β-arrestin mobilization assay. The assay was performed in the presence and absence of HSA to estimate the effect of HSA binding to Nanobodies during the assay. A longer linker preceding the ALB8 building block was evaluated to minimize steric interference of HSA binding to the Nanobody (Table B-14).

Example 17: Characterization of untagged Nanobodies To exclude any effects of Tag-3 on the efficacy of Nanobodies, selected Nanobodies were expressed without Tag-3, β-arrestin mobilization assay and SDF- Both in one competing FACS were characterized in the presence of 2 mg / ml HSA (as further substantially described in Example 10) and efficacy was assessed (Table B-15 and FIG. 8). “Group 2 ISVD” —constructs comprising “Group 2 ISVD” (eg, represented by clone 086) and “Group 2 ISVD” —constructs comprising “Group 1 ISVD” (eg, represented by clone 085) Is more effective at replacing SDF-1 than a construct comprising “Group 1 ISVD” — “Group 1 ISVD” (eg, represented by clone 093). Competition with a construct comprising “Group 1 ISVD” — “Group 1 ISVD” (eg, represented by clone 093) is less effective.

  These data support the radioligand competition assay in which the monovalent 01C10 was tested (see Table B-10: 31% for 01C10). Thus, Group 2 ISVDs are excellent SDF-1 substitutes.

Example 18: Immunohistochemical analysis of CXCR7 expression in primary tumor sections

  Tumor sections analyzed for CXCR7 expression were derived from a human primary tumor of variable cancer type that was passaged once in nude mice. Paraffin-embedded tumors were cut into 5 μm sections (using a Leica RM 2135 microtome), dried, dewaxed, and stained with hematoxylin and eosin. A representative area could then be marked on these tumor sections and a 1 mM diameter cone could be punched to assemble a Tissue Micro Array (TMA). TMA was then prepared according to Mirlacher and Storz using a Beecher Instruments Micro Tissuearrayer (Mirlacher M. and Storz M., 2000, Gewebe-Chips fur die molekulare Untersuchung von Tumoren, Labmed., 293-297). Array sections were cut using the Instrumedics Sectioning Aid System and specifically coated using “Starfrost” slides.

Immunohistochemical staining of CXCR7 expressing tissue was performed as follows: (1) Paraffin was removed from the tissue, tissue was dehydrated and washed; (2) Endogenous peroxidase was removed from 3% H in distilled water. Inactivated by addition of ₂ O ₂ ; (3) Specimens were dried upon washing; (4) Non-specific binding was blocked by 10% BSA in PBS; (5) Anti-human / mouse CXCR7 monoclonal Antibody (Biolegend, clone Mab 8F11) or isotope control antibody (Biolegend, IgG2b) was incubated at a concentration of 25 μg / mL, then the tissue was washed; (6) Second antibody goat anti-mouse biotinylated IgG (Jackson ImmunoResearch) Incubated at a final concentration of 2.8 μg / mL and the tissue was subsequently washed; Performed with peroxidase substrate, each step, a washing step; (8) counter-stained and (9) using hematoxylin continued dehydration of the tissue.

  TMA (170 tumor models) was evaluated semi-quantitatively using a Zeiss Axiovert 35 microscope. Pictures were taken using a Zeiss AxioCam MRc camera. All tumor samples were evaluated in duplicate. Staining was interpreted based on the percentage of cells that stained positive as well as signal intensity. Samples were grouped into the following categories: 0, no staining (no antigen present); 1, weak staining; 2, moderate staining; 3, strong staining.

  FIG. 3 gives an overview of the scores assigned to the different tumor types. High CXCR7 expression (score = 3) in at least one of the two tissue patches was found in 55 (= 32.4%) of the 170 tumors tested. Nine tumors did not show CXCR7 expression (staining score = 0). Weak or intermediate expression (score 1 and 2) was found for the remainder of the xenograft tissue. Notably, the majority of colon cancer tumors (19/23 or 82.6%) and gastric cancer tumors (8/12 or 66.7%) showed only no staining or weak staining (score ≦ 1) In contrast, all of the head and neck tumors tested (7/7 or 100%) showed relatively high CXCR7 expression (score ≧ 2). In other tissue types, however, CXCR7 staining was highly variable between individual tumor models.

  For some tumor samples, the staining intensity was confirmed on all tumor sections.

Example 19: CXCR7 Nanobodies Reduce Tumor Growth in Head and Neck Cancer Xenografts In Vivo 19.1 Materials and Methods 19.1.1 Cell Line Cell Line UM-SCC-11B (11B) After receiving chemotherapy, it was cultured from a biopsy of primary laryngeal cancer. The cell line UM-SCC-22A (22A) was derived from primary squamous cell carcinoma of the oropharynx. The cell line UM-SCC-22B (22B) was derived from oropharyngeal metastatic squamous cell carcinoma. Human head and neck squamous cell carcinoma (HNSCC) cell lines FaDu and HNX-OE have been described earlier (Hermsen et al., (1996) "Centromeric breakage as a major cause of cytogenetic abnormalities in oral squamous cell carcinoma" Genes Chromosomes Cancer 15: 1-9; Ranger (1972) "A new human cell line (FaDu) from a hypopharyngeal carcinoma" Cancer 29: 117-121). The HNX-OE and 93-VU-147T cell lines were established at Vrije Universiteit Amsterdam (Hermsen et al. Ibid), whereas the FaDu strain was obtained from Karl-Heinz Heider (Boehringer Ingelheim Austria).

19.1.2 Quantitative RT-PCR analysis Total RNA was extracted from head and neck cancer cell lines using the RNeasy kit from Qiagen according to the manufacturer's protocol. Messenger (m) RNA was converted to cDNA using the BioRad iScript cDNA synthesis kit. Subsequently, mRNA expression levels were detected using CXCR7 and β-actin specific primers from Origene using SyberGreen (BioRad). CXCR7 expression levels were normalized to that of β-actin, allowing comparison of different cell lines.

19.1.3 Radioligand-conjugated head and neck cancer cell lines were seeded on poly L lysine coated 96 well plates and grown overnight. The next day, 0.5% BSA was added to the binding buffer (50 mM Hepes pH 7.4, 1 mM CaCl ₂ , 5 mM MgCl ₂ , 0.1 M NaCl) and chemokine (10 ⁻⁷ M) or CXCR7 specific Added to cells in the absence or presence of any of Nanobodies 9A4 (10 ⁻⁶ M). Thereafter, radiolabeled [ ¹²⁵ I] -CXCL12 (Perkin-Elmer) was added to reach a final concentration of 75 pM. Cells were incubated for 3 hours at 4 ° C. and washed twice with binding buffer containing 0.5M NaCl. Samples were collected using lysis buffer and the remaining cell-bound radioactivity was counted.

19.1.4 Animal Experiments All animal experiments are conducted in accordance with NIH principles of laboratory animal care and the Dutch national legislation ["Wet op de Dierproeven" (Stb 1985, 336)] and approved by Dierexperimentencommissie from VU University Medical Center. This was carried out according to protocol FaCh 10-01. Head and neck cancer cells 22A were injected subcutaneously into the flank of 8-10 week old female donor nude mice (Hsd, athymic nu / nu, Harlan laboratories). Xenograft tumors were grown to a size of 200-500 mm ³ and then excised, cut into equal sized pieces and implanted subcutaneously into the flank of recipient nude mice. If the transplanted tumors engrafted correctly, the mice were injected intraperitoneally every other week with PBS or either 1 mg divalent nanobodies or 1.5 mg trivalent nanobodies.

19.2 Results 19.2.1 mRNA expression of CXCR7

  Head and neck cancer cell lines were first tested for CXCR7 mRNA expression. Of the 6 cell lines tested, 4 cell lines showed CXCR7 mRNA expression (ie, 22A, 22B, OE, and 93-VU-147 cell lines) (FIG. 4).

19.2.2 Protein expression of CXCR7 CXCR7 mRNA is expressed in a wide range of tissues in humans. However, mRNA expression does not always correlate with protein cell surface expression. Therefore, to further assess the presence of CXCR7 protein, protein expression of CXCR7 was confirmed in a [ ^125I ] -CXCL12 radioligand binding assay. CXCR7-specific expression was determined by replacing the radioligand with the cold chemokines CXCL12 and CXCL11 (but not CXCL10). In addition, monovalent Nanobody 09A04 replaced [ ¹²⁵ I] -CXCL12 to the same extent as CXCL11 and CXCL12 (FIG. 5).

  These data confirmed that mRNA and protein CXCR7 are expressed in four head and neck cancer cell lines.

19.2.3 CXCR7 Nanobodies used CXCR7 expressing cell lines that can inhibit tumor growth in vivo in a xenograft model, where tumor growth was measured. The 22A cell line was chosen as the xenograft tumor model. This is because nude mice injected subcutaneously with 2 × 10 6 cells per flank allowed xenograft tumor formation. Next, we performed tumor transplantation to ensure that mice from different groups (treated vs. untreated) presented similar initial tumor sizes for therapeutic experiments. Initially, donor nude mice were initially injected subcutaneously in their flank with 2 × 10 ⁶ 22A cells. Tumors were grown to a size of 200-500 mm ³ and then removed, cut into equal sized pieces and implanted subcutaneously in recipient nude mice. When engrafted tumors began to grow, mice were randomly distributed into 5 groups, and they were injected biweekly with 400 μl PBS with or without Nanobodies. The constructs tested for treatment are clone 060, clone 083, clone 085 and clone 093. Bivalent and trivalent Nanobodies were administered at 1 and 1.5 mg / injection, respectively. Over the 50 day treatment period, the control (PBS) group and the clone 060 and clone 083 groups grew tumors to a similar extent (no significantly different sizes) (FIG. 6). Mice treated with clone 085 and clone 093 exhibited slower tumor growth and significantly smaller size compared to PBS injected mice at the end of the treatment experiment (tumor volume PBS = 274 ± 47 mm ³ , clone). 085 = 119 ± 30 mm ³ and clone 093 = 114 ± 32 mm ³ ) (FIG. 7).

  Thus, CXCR7 Nanobodies reduce head and neck cancer cell growth in vivo.

  This study not only supports the anti-tumor efficacy of Nanobodies, but also supports an excellent safety profile (reflecting its highly targeted specific activity profile), which is basically in development Or unlike many other cytotoxic drugs on the market.

Example 20: CXCR7 Nanobodies Reduce Xenograft Tumor Growth In Vivo In Example 19, it has been demonstrated that CXCR7 Nanobodies can inhibit tumors, as exemplified by head and neck cancer.

  Additional xenograft models can be used in the first phase, which demonstrates in vivo that Nanobodies are also more effective than head and neck cancer in other tumors where (over) express CXCR7. Glioma is the most common form of primary human brain tumor, and they are often classified into four clinical grades. The most invasive tumor (grade 4 tumor), also known as glioblastoma multiforme (GBM), is associated with high mortality and morbidity. The survival of patients affected by GBM has remained substantially unchanged for the past decades despite advances in surgery, radiation, and chemotherapy (ie, 6-12 months after diagnosis). . GBM xenograft models are substantially described by, for example, Yi et al. (EGFR Gene Overexpression Retained in an Invasive Xenograft Model by Solid Orthotopic Transplantation of Human Glioblastoma Multiforme Into Nude Mice "Cancer Invest. 2011 29: 229-239). Can be used as is.

  In effect, a xenograft set up as described in Example 19 is used, but a xenograft derived from the primary tumor is used, which is obtained from a patient who has undergone a surgical procedure. Cells from these tumors are injected into 4-6 week old congenital athymic nude mice (female, Balb / c nu / nu background). Mice are maintained in a barrier environment of specific pathogen removal. For implantation and imaging, mice are anesthetized intraperitoneally with 0.10 mg ketamine hydrochloride solution per gram body weight. If necessary, tumors are excised and re-implanted into other mice as described in Example 19.

  Treatment is initiated with biweekly injections of either 1.5 mg PBS, clone 060, clone 083, clone 085, and clone 093. Tumor size is measured every 4 days. Tumor size is measured with calipers and tumor volume is calculated using the formula (length x width 2) / 2. The development of mouse tumor volume is followed over 30 days. On day 30, the mice are sacrificed. Tumors are weighed and fixed in 4% polyformaldehyde. Tumor sections are excised for immunohistochemical analysis.

  The tumors listed in FIG. 3 are similarly tested with xenografts from established cell lines or from primary tumors. Tumors with a high percentage of CXCR7 are preferred for initial testing.

Example 21: Single variable domain of group 1 immunoglobulins In view of binding, competition, and / or beta arrestin results, various ISVDs were not further evaluated after initial screening. However, the in vivo results of Examples 19-20 prompted the inventors to further evaluate the presence of other family members of Group 1 ISVD.

  After sequence evaluation, at least the following four Group 1 ISVDs were identified: 01C12 (SEQ ID NO: 99), 01B12 (SEQ ID NO: 100), 01F11 (SEQ ID NO: 101), and 01B10 (SEQ ID NO: 102) (Table B -3).

Example 22: CXCR7 Nanobody Reduces Head and Neck Cancer Xenograft Tumor Growth In Vivo In Example 19, CXCR7 Nanobody was demonstrated to reduce head and neck cancer cell growth in vivo. In Example 19, mice received either 1.5 mg of clone 085 (Group 1 ISVD—Group 2 ISVD) or clone 093 (Group 1 ISVD—Group 1 ISVD).

  In view of the binding efficacy of Group 2 ISVD, constructs containing Group 1 ISVD-Group 2 ISVD (eg, clone 085) are more efficient than Group 1 ISVD-Group 1 ISVD (eg, clone 093). Expected to be

  Therefore, this hypothesis is tested using the in vivo xenograft model of Example 19. Again, mice were randomly distributed into 11 groups of 5 each, and they were injected biweekly with 400 μl PBS with or without construct. The constructs tested for treatment are clone 085 and clone 093.

Administration follows the following scheme:

  Tumor size is measured every 4 days. Tumor size is measured with calipers and tumor volume is calculated using the formula (length x width 2) / 2. The development of mouse tumor volume is followed over 30 days. On day 50, the mice are sacrificed. Tumors are weighed and fixed in 4% polyformaldehyde. Tumor sections are excised for immunohistochemical analysis.

Claims (35)

  A single variable domain of amino acid residue M33 in CXCR7 (SEQ ID NO: 1) and optionally at least one immunoglobulin that binds to and / or recognizes amino acid residue V32 and / or amino acid residue M37 ( ISVD) and amino acid residue WF19 of CXCR7 (SEQ ID NO: 1) and optionally a construct comprising at least one ISVD that binds to and / or recognizes S23 and / or D25.   The construct according to claim 1 for use as a medicament for reducing tumor growth and / or treating cancer, preferably head and neck cancer or GBM.   A single immunoglobulin that can specifically substitute SDF-1 and I-TAC on human CXCR7 (SEQ ID NO: 1) with an average Ki of less than 100 nM and an average SDF-1 and I-TAC substitution of 50% or more. One variable domain.   A single variable domain of an immunoglobulin capable of specifically replacing SDF-1 on human CXCR7 (SEQ ID NO: 1) with an average Ki of less than 100 nM and an average SDF-1 substitution of 50% or more.   A single variable domain of an immunoglobulin capable of specifically substituting I-TAC on human CXCR7 (SEQ ID NO: 1) with an average Ki of less than 100 nM and an average I-TAC substitution of 50% or more.   The single variable domain of an immunoglobulin according to any one of claims 3 to 5, wherein the average Ki is 50 nM or less.   The single variable domain of an immunoglobulin according to any one of claims 3 to 5, wherein the average Ki is 10 nM or less.   The single variable domain of an immunoglobulin according to any of claims 3 to 7, wherein the mean SDF-1 or I-TAC substitution is 80% or more.   A single variable domain of an immunoglobulin capable of binding to human CXCR7 (SEQ ID NO: 1) with a Kd of less than 50 nM.   A single variable domain of an immunoglobulin that binds to and / or recognizes amino acid residue M33 in CXCR7 (SEQ ID NO: 1), and optionally amino acid residue V32 and / or amino acid residue M37.   A single variable domain of an immunoglobulin that binds to and / or recognizes amino acid residue WF19 of CXCR7 (SEQ ID NO: 1) and optionally S23 and / or D25.   12. An immunoglobulin single variable domain according to claim 10 or 11 for use as a medicament for reducing tumor growth and / or treating cancer, preferably head and neck cancer or GBM. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 9;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 9;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 9; and
Here, CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 19;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 19;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 19; and
Here, CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 29;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 29;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 29. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 10;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 10;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 10; and
Here, CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 20;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 20;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 20; and
Here, CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 30;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 30;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 30. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 11;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 11;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 11;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 21;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 21;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 21;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 31;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 31;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 31. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 12;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 12;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 12;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 22;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 22;
f) a single variable domain of an immunoglobulin having a 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 22;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of an immunoglobulin of SEQ ID NO: 32;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 32;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 32. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 13;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 13;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 13;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 23;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 23;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 23;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 33;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 33;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 33. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) the single variable domain of an immunoglobulin of SEQ ID NO: 93;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 93;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 93;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 95;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 95;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 95;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of an immunoglobulin of SEQ ID NO: 97;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 97;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 97. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 107;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 107;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 107;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 115;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 115;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 115;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 123;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 123;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 123. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) a single variable domain of the immunoglobulin of SEQ ID NO: 108;
b) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 108;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 108;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 116;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 116;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 116;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 124;
h) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 124;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 124. The immunoglobulin single variable domain of any of claims 3-12, wherein the immunoglobulin single variable domain is of the formula 1
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1)
An amino acid sequence with
Where FR1-FR4 refers to framework regions 1-4 and is a framework region of an immunoglobulin single variable domain; and
Here, CDR1 is selected from the group consisting of:
a) the single variable domain of the immunoglobulin of SEQ ID NO: 110;
b) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 110;
c) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 110;
And where CDR2 is selected from the group consisting of:
d) the single variable domain of the immunoglobulin of SEQ ID NO: 118;
e) a single variable domain of an immunoglobulin having at least 80% amino acid identity with the single variable domain of the immunoglobulin of SEQ ID NO: 118;
f) a single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 118;
And where CDR3 is selected from the group consisting of:
g) the single variable domain of the immunoglobulin of SEQ ID NO: 126;
h) an immunoglobulin single variable domain having at least 80% amino acid identity with the immunoglobulin single variable domain of SEQ ID NO: 126;
i) A single variable domain of an immunoglobulin having 3, 2, or 1 amino acid difference from the single variable domain of the immunoglobulin of SEQ ID NO: 126.   The framework region (FR) is SEQ ID NO: 4-8, 92, 103, 104, or 106 (FR1), 14-18, 94, 111, 112, or 114 (FR2), 24-28, 96, 119, 22. A sequence identity greater than 80% with a FR of 120 or 122 (FR3) and / or 34-38, 98, 127, 128, or 130 (FR4). A single variable domain of an immunoglobulin.   23. A polypeptide comprising a single variable domain of an immunoglobulin according to any of claims 3-22.   An immunoglobulin single variable domain is an immunoglobulin single variable domain having an amino acid sequence with greater than 80% sequence identity with an immunoglobulin single variable domain of SEQ ID NOs: 39-43, 91, or 99-102 24. The polypeptide of claim 23, selected from the group consisting of:   25. Any of claims 23 to 24, further comprising a single variable domain of at least one human serum albumin binding immunoglobulin, optionally comprising a linker selected from the group of linkers with SEQ ID NOs: 49-58. The described polypeptide.   In addition, a polypeptide according to any of claims 23 to 25, comprising a linker comprising ALB8 (SEQ ID NO: 2) and optionally selected from the group of linkers with SEQ ID NO: 49-58.   27. The polypeptide is selected from the group consisting of polypeptides having an amino acid sequence with greater than 80% sequence identity with the polypeptides of SEQ ID NOs: 44-48, 78-89, and 131-140. The polypeptide according to any one of the above. A construct selected from the group consisting of:
A construct comprising amino acid residue WF19 of CXCR7 (SEQ ID NO: 1) and optionally at least two ISVDs that bind to and / or recognize S23 and / or D25, wherein said at least The two ISVDs can be the same or different;
A construct comprising amino acid residue M33 in CXCR7 (SEQ ID NO: 1) and optionally at least two ISVDs that bind to and / or recognize amino acid residue V32 and / or amino acid residue M37, Where the at least two ISVDs can be the same or different;
A construct comprising at least one group 1 ISVD and at least one group 2 ISVD;
A construct comprising at least one group 1 ISVD and at least one group 3 ISVD;
-A construct comprising at least one group 2 ISVD and at least one group 3 ISVD; and-a construct comprising at least one 01C10-like sequence and at least one 14G03-like sequence.   29. A construct according to claim 28 for use as a medicament for reducing tumor growth and / or treating cancer, preferably head and neck cancer or GBM. A nucleic acid sequence encoding: i) a single variable domain of an immunoglobulin according to any of claims 3 to 22;
ii) a polypeptide according to any of claims 23 to 27; or iii) a construct according to any of claims 1, 2, 28 or 29. A pharmaceutical composition comprising i) an immunoglobulin single variable domain according to any of claims 3 to 22;
ii) a polypeptide according to any of claims 23 to 27; or iii) a construct according to any of claims 1, 2, 28 or 29;
And optionally a pharmaceutically acceptable excipient.   A single variable domain of an immunoglobulin according to any of claims 3 to 22, a polypeptide according to any of claims 23 to 27, or cancer, preferably head and neck cancer, GBM, and / or inflammatory disease 30. A construct according to any of claims 1, 2, 28, or 29 for use in.   A single variable domain of an immunoglobulin according to any of claims 3 to 22, a polypeptide according to any of claims 23 to 27, or claim 1, 2, 28, or for use in rheumatoid arthritis. The construct according to any one of 29.   A single variable domain of an immunoglobulin according to any of claims 3 to 22, a polypeptide according to any of claims 23 to 27, or claims 1, 2, 28 for use in multiple sclerosis. Or the construct according to any one of 29. 30. The immunoglobulin single variable domain of any of claims 3-22, the polypeptide of any of claims 23-27, or of any of claims 1, 2, 28, or 29. A method for producing a construct, the method comprising at least the following steps:
a) expressing the nucleic acid or nucleotide sequence of claim 30 in a suitable host cell or host organism or in another suitable expression system; optionally followed by:
b) a single variable domain of an immunoglobulin according to any of claims 3 to 22, a polypeptide according to any of claims 23 to 27, or any of claims 1, 2, 28 or 29; Isolating and / or purifying the described construct.